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FRET-based nanobiosensor for detection of scopolamine in hairy root extraction of Atropa belladonna
Author’s Accepted Manuscript FRET-based nanobiosensor for detection of scopolamine in hairy root extraction of Atropa belladonna Fereshte Bagheri, Khosro Piri, Afshine Mohsenifar, Smaiil Ghaderi www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(16)30954-7 http://dx.doi.org/10.1016/j.talanta.2016.12.013 TAL17103
To appear in: Talanta Received date: 27 May 2016 Revised date: 2 December 2016 Accepted date: 6 December 2016 Cite this article as: Fereshte Bagheri, Khosro Piri, Afshine Mohsenifar and Smaiil Ghaderi, FRET-based nanobiosensor for detection of scopolamine in hairy root extraction of Atropa belladonna, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.12.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
FRET-based nanobiosensor for detection of scopolamine in hairy root extraction of Atropa belladonna Fereshte Bagheri 1, 21, Khosro Piri 1,*2, Afshine Mohsenifar 33, Smaiil Ghaderi 4 1
2
Department of Biotechnology, College of Agriculture, Bu-Ali Sina University, Hamedan, Iran Research pharmaceutical center, School of pharmacy, Kermanshah University of Medical Sciences,
Kermanshah, Iran 3
4
Research and Development Department of Nanozino, Tehran, Iran Department of Chemistry. Bu-Ali Sina University, Hamedan, Iran
[email protected] [email protected] [email protected] * Author to whom correspondence should be addressed; Tel.: +98 918-813-0783; Fax: +9821-82233470.
External Editor:
1
Speciality: Biosensor- Biotechnology- Drug Delivery Systems, Academic Status: Researcher, MS: Biotechnology, BS/BA: Agriculture engineering, Adress: Research pharmaceutical center, School of pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran 2 Speciality: Biotechnology, Academic status: Assistant professor 3 Speciality: Biosensors - Drug Delivery Systems, Academic Status: Assistant Professor, PhD: Clinical Biochemistry , MS: Clinical Biochemistry , BS/BA: Laboratory Sciences, Address: Research & Development Department of Nanozino, Tehran, Iran. Phone: 021-77061120; Fax: 021-44787371
ABSTRACT A simple, sensitive, selective, and rapid optical nanobiosensor based on FRET was designed to detect tropane alkaloids as anti-cholinergic agents in natural and transgenic hairy roots extracts of Atropa belladonna. To achieve that, conjugation of tioglycolyic acid capped cadmium telluride quantum Dots, M2 muscarinic receptor (Cd/Te QDs-M2R) and conjugation of scopolamine-rhodamine123 (Sc-Rho123) were performed. More specifically, proportional amounts of M2 muscarinic receptor and quantum dots (QDs) were conjugated while scopolamine (as a tropane alkaloid) and rhodamine123 were also combined and these moieties functioned as donor and acceptor pairs, respectively. The system response was linear over the range of 0.01-4 µmol.L-1 of scopolamine hydrochloride concentration with a detection limit of 0.001 µmol.L-1. The developed nanobiosensor was successfully used for in vitro recognition of scopolamine as an anti-cholinergic agent in the investigated plant extracts. In addition, Agrobacterium rhizogenesis mediated gene transfer technique was employed to generate hairy roots and to enhance the production of tropane alkaloids in the studied medicinal plant.
Graphical Abstract
Construction of CdTeTGA-M2-ScRhod complex by combination of CdTe-TGA-M2, ScRhod conjugation and FRET system between two fluorophores, CdTe and rhodamine123 established.
Keywords: Nanobiosensor; FRET; Cd/Te Quantum Dots; M2 muscarinic receptor; Tropane alkaloids; Hairy roots
1. Introduction Nanoparticles are of considerable interest in the biotechnological field owing to their wide applications in drug delivery, in vitro and in vivo monitoring in cellular research, nucleic acid analysis, and signal transduction. Moreover, their unique electronic and magnetic properties have marked them as excellent materials for designing and developing sensors and biosensors [1]. Among various types of nanobiosensors, FRET-based ones due to their rapid and highly sensitive mechanisms have attracted a lot of attention for quantification of different target analyts. In fact, FRET-based nanobiosensors are considered as powerful instruments offering specific applications, e.g., in vitro and in vivo sensing of protein- protein interactions, receptor/ligand interactions, medicinal diagnostics, estimating of DNA/RNA concentration as well as in cancer research. It is worth quoting that FRET-based
nanobiosensors have greatly simplified experimental measurements owing to the fact that their quantitative assays are easily performed [2- 5]. The emergence of semiconductor quantum dots (QDs) and their combination with FRET have greatly enhanced the application of this technology within the field of biotechnology. In fact, recent years have witnessed a fast growth in the number and diversity of QDs nanoparticles, FRET-based biosensors developed [4]. QDs nanoparticles are fluorescent materials which illustrate excellent photo-physical properties such as high brightness and photostability. Among the II-IV semiconductors, Cd/Te QDs nanoparticles are considered as promising material for various biological applications owing to their attractive features, i.e., broad excitation spectrum, narrow emission spectrum, fluorescent quantum yields, facile synthetic versatility and having a direct band gap as well as bulk band gap energy of 1.48 eV which are suitable for sensors designing. Hence, the uses of Cd/Te QDs have been particularly advantageous in the field of biosensors development [5]. Various stabilizers have also been used in the synthesis of QDs [4- 6]. Among which, many sensors have been established based on the surface interaction of thioglycolic acid and a specific detecting moiety. Therefore, functionalized QDs have been introduced as one of the most important fluorescent sensors for many analyts. In fact, expanding the application of the capped QDs to develop fluorescent sensors in aqueous media is a topic of current interest. Earlier studies related to the interactions among QDs and many analyts have revealed that surface capping ligands have noticeable effects on the fluorescence response of QDs to physiologically
important
biomolecules
such
as
ions,
small
molecules
and
biomacromolecules [6]. Anticholinergics are a class of drugs used for treating diseases like asthma, incontinence, gastrointestinal cramps, and muscular spasms [2]. They are also prescribed for depression and sleep disorders [3]. Their action mechanism involves the blocking of the
neurotransmitter acetylcholine in the brain [7]. Tropane alkaloids as anticholinergic and pharmaceutical agents can competitively bind to the neurotransmitter, acetylcholine, in the central nervous system. Their targets are either muscarinic acetylcholine receptors or, less commonly, nicotinic acetylcholine receptors [8]. Atropa belladonna plant is a very rich source of tropane alkaloids such as scopolamine, hyocsyiamine and its racemic form atropine (Fig.1) [9]. It is well-documented that genetic modifications can increase the production of metabolites of interest. In fact, rapid growth of root cultures and significant increase of many secondary metabolites can be established either by manipulation of auxin levels in the culture medium or by genetic transformation of plants with Agrobacterium rhizogenesis [10-11]. A. rhizogenes is able to transform plant cells by introducing a part of its Ri-plasmid (T-DNA) into the plant genome [12-13]. Pharmaceutical applications of tropane alkaloids have led to an interest for precise determination of these active substances in medicinal plants. Various analytical methods such as TLC, HPLC, GC, GC/MS and LC-MS have been reportedly employed for the detection and quantification of tropane alkaloids in medicinal plants [14]. Furthermore, detection of tropane alkaloids especially scopolamine and atropine in pharmaceutical formulations have also accomplished using potentiometric sensor [15], atropine sulfate bulk acoustic wave sensor [16], LC-MS-MS [17], electro-chemiluminescance electrospun carbon nanofiber based sensor [18], as well as β-cyclodextrin modified ion sensitive field effect transistor sensor [19]. However, all these methods are expensive and time-consuming. On the contrary, nanobiosensor systems are novel, sensitive, selective, fast, reliable, and simple. More specifically and as mentioned earlier, FRET-based nanobiosensors have been known for their superior sensitivity and applicability for negligible volumes of specific pharmaceuticals [20]. Nevertheless, to the best of our knowledge, the use of functionalized Cd/Te QDs as Förster resonance sensor for selective recognition of anticholinergic drugs is
virtually unexplored. This is the first report on quantification analysis of tropane alkaloids in natural and hairy root extracts by using a FRET-based nanobiosensor (Fig. 2). To achieve that, M2 muscarinic receptor was applied as a biorecognition element. It is worth noting that M2 muscarinic receptor (M2R) belonging to G protein-coupled receptors (GPCRs) has substantial roles in both central and parasympathetic nervous systems. This receptor acts as an essential protein for physiological control of cardiovascular function [21]. TGA-capped Cd/Te QDs nanoparticles were synthesized and conjugated to M2 muscarinic receptor as donor. On the other hand, scopolamine-Rho123 conjugation was prepared as acceptor. The synthesized conjugates were then used in a FRET-based nanobiosensor system for the detection of tropane alkaloids in the natural extract and the hairy root extract of A. belladonna transformed by A. rhizogenesis, AR15834 strain. Suggested place for Fig. 1
Suggested place for Fig. 2 2. Experimental 2.1. Reagents Seeds of A. belladonna were collected from a medicinal plants garden located in Hamadan, Iran. A. rhizogenesis, AR15834 strain, was purchased from the Biotechnology Research Center, Karaj, Iran. Scopolamine hydrochloride, Rho123, CdCl2.2.5H2O, NaBH4, Tellurium
powder,
(TGA),
tetramethylammonium
hydroxide,
1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma (Germany). M2 muscarinic receptor was obtained from Jackson Immuno research Laboratory (USA). All the other chemicals used were purchased from Merck (Germany). 2.2. Experimental apparatuses
DNA amplification was performed by using a Bio-Rad thermo cycler (USA). FT-IR analysis was performed on a Perkin Elmer FT-IR spectrum (USA). The transmission electron microscopy (TEM) image was obtained by using a Philips H600 (Netherland). All fluorescence spectra were recorded by Perkin-Elmer LS-50B fluorescence Spectrometer (USA).
2. 3. Oxidation of primary alcohol of scopolamine In order to attach scopolamine to Rho123, the primary alcohol of scopolamine oxidized to the aldehyde. Barium manganate (BaMnO4), an oxidizing agent can use as a very effective catalyst for selective oxidation of primary alcohols to aldehydes [22]. Briefly, 102 mg of scopolamine was dissolved into 40 ml of chloroform and then 1.5 g of BaMnO4 was added to the mixture. The reaction mixture was kept under magnetic stirring and reflux for 20 min. Reaction progression was monitored by a TLC in petroleum ether/chloroform (50:50 v/v). Subsequently, the mixture was dried over anhydrous Na2SO4, and filtered with chloroform. Finally, the solvent was evaporated and the dried product was purified by a TLC plate in petroleum ether/chloroform (50:50 v/v).
2.4. Conjugation of scopolamine to Rho123 After scopolamine was oxidized to its corresponding aldehyde, it was conjugated to Rho123 through the primary amine of the dye as described previously by Reddy Chereddy [23] with some modifications. In brief, under reflux and N2 bubbling condition, 10 mg of Rho123 was dissolved in 30 ml of methanol, and then 10 mg the oxidized scopolamine was added slowly. The mixture was agitated at 60°C for 6 h to yield a dark purple solution. Furthermore, the progress of reaction was monitored by TLC. Then, the mixture was cooled at room temperature. Subsequently, it was filtered and washed 3 times with methanol. The
resulting solution was dried at room temperature. Finally, the FT-IR spectrum of the generated product was taken to prove imine bound formation.
2.5. Preparation of TGA-capped Cd/Te QDs Colloidal TGA-capped Cd/Te QDs were prepared according to the literature [24- 25]. In brief, 100 mg of Te powder was reduced by 280 mg of NaBH4 in 7 ml of double-distilled water under N2 atmosphere and magnetic stirring. After 5 h, the color of the obtained solution (NaHTe) was changed from violet to white. On the other hand, 358 mg CdCl2.2.5 H2O and 0.2 ml of TGA were dissolved in 200 mL deionized water and the pH of the solution was adjusted to 10 by drop-wise addition of NaOH solution (1 M). The produced Cd2+ solution was then de-aerated under nitrogen bubbling. Subsequently, the H2Te gas was obtained by reacting the NaHTe solution with diluted H2SO4 (0.5 mol.L-1) and was then passed through the oxygen-free Cd2+ solution with a gentle nitrogen flow (under stirring condition). The mixture was refluxed under nitrogen atmosphere together with sever stirring. By prolonging the refluxing time, different sizes of QDs was achieved. In order to eliminate excess precursors, the crude solution was washed three times with ethanol and centrifuged at 10,000×g for 15 min. Then, the precipitate was re-dispersed in 250 mL of water and kept at 4°C in darkness. Maximum excitation and emission wavelengths were 375 and 505 nm, respectively.
2.6. Conjugation of TGA-capped Cd/Te QDs and M2 muscarinic receptor Since the synthesis of the Cd/Te QDs was performed in an aqueous solution and owing to carboxylic group of the capping agent, the surface of the QDs was negatively charged. For conjugating the QDs to M2 muscarinic receptor, firstly, 1ml of the prepared QDs solution was mixed in to 5 ml of EDC (1 mg) and NHS (1 mg) solution at pH 6 and was
incubated in darkness for 1h at room temperature to ensure the attachment of EDC and NHS onto the surface of the QDs. Then, appropriate mounts of an ice-chilled solution of M2 muscarinic receptor was added to the activated QDs solution under mild magnetic stirring and this condition was maintained for 1h. Therefore, positively charged M2 muscarinic receptor interacted with the QDs through charge transfer mechanism. The nanobioconjugate stock solution was stored at 4°C in darkness until use.
2.7. Construction of the QDs-M2R/Sc-Rho123 To verify the formation of QDs-M2R/ScR- Rho123 complex, 2 ml of phosphate buffer (pH 7, 50 mM) and 0.5 mL of the QDs-M2R complex solution (as donor) were initially introduced into a fluorimeter cell and subsequently 0.5 mL of the Sc-Rho123 complex solution (as acceptor) was added to the reaction mixture. After incubation for 3 min, successful construction of the QDs-M2R/Sc-Rho123 complex was investigated through spectrofluorometry (excitation at 350 nm; 25 nm bandwidth for both excitation and emission spectra). 2.8. Hairy root induction Seeds of A. belladonna were cultured on 1/2MS medium. Then, the pieces of leaves, stems, and roots of 14-28 days plantlets were infected by A. rhizogenesis, AR15834 strain, containing Ri-plasmid. The plantlets inoculated with the Agrobacterium were cultured in 1/2MS medium containing cefotaxime (500 mg.L-1). After 7-10 days, hairy roots of A. belladonna were observed. The hairy roots were sub-cultured in 2 weeks intervals. Regeneration of transgenic plants from hairy roots can be either spontaneous or can be induced with the help of plant growth regulators. In the present study, spontaneous plantlets regeneration was observed in hairy root cultures after 3 sub-culturing passages. Plantlets regeneration was optimized in a greenhouse as elaborated [26]. Hairy roots, leaves, and stems
of the regenerated plants were extracted using the method described by Kamada et al. [27]. All samples were kept at 4°C and were used for detection of tropane alkaloids by using the designed nanobiosensor system. 2.9. DNA analysis To ensure the formation of transgenic hairy roots and the regenerated plantlet, DNA was extracted using the CTAB method [28]. PCR analysis was performed using specific primers to confirm the presence of the rol B gene in the hairy root, leaves, and stems of the regenerated plantlets. Natural roots and the plasmid A. rhizogenesis were used as negative and positive controls, respectively.
2.10. Evaluation of nanobiosensor for assessment of tropane alkaloids in plant extracts Following the optimization of the developed nanobiosensor and for its assessment, a solution including 2 ml of phosphate buffer (50 mM, pH 7), 50 μl of the QDs-M2R nanobioconjugate, and 50 μl of the Sc-Rho123 conjugate was added to the detection system. Subsequently, different concentrations of the plant extracts including those of hairy roots, leaves, stems, and the root extract of Datura innoxia (as positive control) and Glycyrrhiza glabra (as negative control) were used for assaying their tropane alkaloids contents (Fig. 7). 3. Result and discussions Tropane alkaloids (scopolamine and hyoscyamine) in hairy roots, plants and other environmental and biological samples have been analyzed by routinely analytical methods such as HPLC [29- 30], GC [31], GC/MS [32- 33], and liquid chromatography-mass spectrometry [34] which as mentioned earlier are expensive and time-consuming. A review of some analytical methods used for quantification of scopolamine in biological samples is presented in Table 1.
Suggested place for Table 1
FRET-based approaches are particularly applied tools for sensing, monitoring, and discovering biological compounds. These systems have been successfully used previously for various applications, e.g., detection of porcine reproductive and respiratory syndrome [41], determination of dopamine [42], detection of bovine serum albumin (BSA) in micelles [43]. Kattke and et al. also applied a similar system for determination of Aspergillus amstelodami [44] while Shanehsaz and et.al developed a FRET-based nanobiosensor for identification of Helicobacter pylori [24]. Here, we report on a novel, sensitive, and simple optical nanobiosensor based on FRET using M2 muscarinic receptor and Cd/Te QDs for quantification of tropane alkaloids in plant extracts.
3.1. Oxidation of scopolamine To construct the nanobiosensor, the primary alcohol scopolamine was oxidized using barium manganate to its corresponding aldehyde. FT-IR spectrum of the purified product was performed for verifying the oxidation of scopolamine to the corresponding aldehyde. The IR spectrum revealed the presence of C=O stretching vibrations at 1731.98 cm-1 and CHO flexural at 2853.89 cm-1 confirming the elimination of OH from scopolamine (Fig. 3). Suggested place for Fig.3
3.2. Sc-Rho123 conjugate formation For preparing the Sc-Rho123 conjugate, the aldehyde group of the scopolamine was bound to the amine agent of rhodamine123, and the imine formation was proved by the FTIR spectrum obtained at the wave length range of 400- 4000 cm-1 (Fig. 4). As displayed, the
intensity of the band at 1650.56 cm-1 was correspond to the imine bond (N=C) indicating the successful attachment of scopolamine to Rho123. Suggested place for Fig. 4
3.3. Characterization of the Cd/Te QDs
The Cd/Te QDs due to their highly wide emission spectrum of high resolution can be applied in sensing many specific quenching materials. Moreover, modification of the outer surface of QDs with anionic carboxylate groups is one of the most commonly applied methods for dispersing QDs in an aqueous solution [4-5]. To achieve that, TGA was used in the present study, followed by the activation of the carboxylic groups of the TGA-capped Cd/Te QDs by EDC/NHS. Absorption and emission wavelengths of the prepared Cd/Te QDs and Rho123 solutions are displayed in Fig. 5. As demonstrated, the results obtained indicated that the maximum emission wavelength of the QDs was at 505 nm, while the maximum absorption and emission wavelengths of Rho123 were at 510 nm and 580 nm, respectively. The full width at half maximum (FWHM) of the QDs emission wavelength was at about 35 nm. Emission spectra of the QDs and absorption wavelength of Rho123 showed maximum spectral overlap which is critical for gaining optimum FRET mechanism. As shown in Fig. 5, the TEM image and DLS analysis of the prepared TGA-capped Cd/Te QDs revealed particles of spherical shape with a particle size of about 3 nm and a good monodispersity. All these attributes indicated the suitability of the QDs for designing the nanobiosensor. At adequately basic pH, the electrostatic repulsion between the QDs maintained a colloidal suspension and a high optical stability without considerable reduction of fluorescence intensity during three months.
Suggested place for Fig. 5
3.4. Cd/Te QDs-M2R nanobioconjugate formation To ensure successful conjugation of the QDs and M2 receptor, the optical density (at 280 nm) of the sample containing the CdTe-M2R before and after centrifugation at 10,000 × g for 20 min at 4°C was recorded by a spectrophotometer. More specifically, the receptor had a typical peak at 280 nm in the absorption wavelength; whose absorbance intensity was reduced after the conjugation between the QDs and M2 receptor took place. This might be attributed to the chemical attachment of the M2 receptor onto the surface of the QDs through the EDC/NHS amidization protocol. In better words, this indicated that the covalent conjugation of the QDs and the M2 receptor was achieved [45- 47].
3.5. Transformation of leaves, stems, and root disks of A. belladonna by A. rhizogenesis and regeneration of hairy root The hairy root was induced in leaves, stems, and root disks of A. belladonna by A. rhizogenesis AR15834 strain. Many altered phenotypic characters, the so-called ‘‘Hairy Root Syndrome’’ can be created due to the combined expression of rol A, rol B, and rol C genes [11]. Rol B gene is associated with reduction in the length of stamens, protruding stigmas, and increase in adventitious roots on stems [10]. A large number of hairy roots were regenerated spontaneously within 6-8 weeks [12-13, 45]. Successful amplification of a 780 bp segment in the present study proved that the roots, leaves, and stems of the regenerated plants were successfully transformed and that they contained rol B oncogene while no amplification was observed for the roots, leaves, and stems of natural plants (Fig. 6).
Suggested place for Fig.6
3.6. FRET assessment FRET is a distance-dependent interaction between the electronic excited states of two dye molecules in which the excitation energy is transferred from a donor molecule to an acceptor molecule without emission of a photon. In fact, it is an important technique for investigating a variety of biological phenomena capable of making changes in molecular proximity. As mentioned above, the QDs-M2R/Sc-Rho123 complex was constructed by mixing of the QDs-M2R and Sc-Rho123 conjugates leading to the occurrence of the FRET phenomenon between the two fluorophores. Fig. 2 shows the occurrence of the FRET system between the two fluorophores (Cd/Te QDs and Rho123). 3.7. Nanobiosensor optimization The optimum excitation and emission spectra of the QDs-M2R conjugate as donor in the FRET system was identified by using its various concentrations The results obtained revealed that the concentration of the QDs-M2R conjugate (150 μmol.L-1) led to the optimum excitation and emission wavelengths of 350 nm and 430 nm, respectively. Moreover, in order to determine the optimal condition of the designed system, the responses of the nanobiosensor to the different amounts of the Sc-Rho123 conjugate (50-300 μmol.L-1) as acceptor was also investigated. At the 150 μmol.L-1 concentration of the QDs-M2R conjugate, the optimum concentration of the Sc-Rho123 conjugate was found at 50 μmol.L-1 leading to the maximum emission spectrum of Rho123 at 568 nm (Fig. 7) In fact, the occurrence of the energy transfer (FRET) between the QDs and the Rho123 in the QDs-M2R/Sc-Rho123 complex was confirmed by measuring the fluorescence intensity in a spectrofluorimeter (Fig. 7). Upon the formation of the QDs-M2R/Sc-Rho123 complex, the FRET phenomenon took place due to the reasonable wavelength overlapping
between the donor and acceptor moieties. It should be noted that QDs have an extended range of absorption wavelengths. Therefore, a vast range of excitation wavelengths was selected. Nevertheless, the excitation wavelength of 350 nm was found suitable in the present study. Suggested place for Fig. 7
3.8. Standard curve Standard curve was plotted for scopolamine hydrochloride by competitively replacing scopolamine hydrochloride at variable concentrations (0.01-4 μmol.L-1) with Sc-Rho123 conjugate at the constant concentration of 50 μmol.L-1 (Fig. 8). As shown, a significant response to scopolamine hydrochloride increasing concentration was observed. More specifically, by increasing scopolamine hydrochloride concentration the fluorescence quenching of Rho123 was intensified. This revealed the accuracy of the constructed nanobiosensor in recognition of scopolamine. The correlation coefficient was recorded at 0.98, with a limit of detection (LOD) of 0.001 µmol.L-1, which was calculated based on the LOD=3S0.K-1 equation, where S0 is the standard deviation of the blank (n=4) and K is the slope of the calibration curve. Suggested place for Fig. 8
3.9. Recognition and measurement of scopolamine in plant extracts Determination of scopolamine in plant extracts was achieved by introduction of various concentrations of the extracts obtained from the hairy root, leaves and stem of A. belladonna and root of D. innuxia to the nanobiosensor (Fig. 9, parts a, b, c, d). Considering the data presented in these graphs, by adding an adequate values of the D. innuxia root extract, the emission peak of Rho123 was decreased. On the contrary, by adding G. glabra extract no changes were observed in the emission peak Rho123 (Fig. 9, part e). In fact, this
was indicative of the specificity and selectivity of the developed nanobiosensor. Finally, scopolamine was quantified in the hairy roots, leaves, and stems of A. belladonna and root of D. innuxia extracts at 0.081, 0.021, 0.001 and 0.01 mg.g-1, respectively. The amount of scopolamine in the hairy roots of A. belladonna, determined by HPLC in our previous report stood at 0.06 mg.g-1 [10-12].
Suggested place for Fig. 9
4. Conclusions A simple optical nanobiosensor based on FRET using M2 muscarinic receptor and Cd/Te ODs was developed herein for the identification/quantification of scopolamine in biological systems. As compared to the other methods reported for the detection and quantification of tropane alkaloids, low detection limit, high sensitivity and selectivity, fast response, and simplicity were readily achieved in the present work. Moreover, the shelf-life and the detection limit of the nanobiosensor were found 6 month and 0.001 µmol. L-1, respectively. The present nanobiosensor can be used in a wide range of fields such as plant metabolites, medicinal research, biomedical diagnostics, and drug discovery. Moreover, it should be highlighted that muscarinic receptors are unique targets for treatment of disease such as Parkinson, Schizophrenia, Alzheimer, chronic obstructive pulmonary diseases [21]. Therefore, the designed nanobiosensor through selective assessment of orthosteric drugs can also assist in treatment of these disorders.
Acknowledgments The authors are grateful to Bu-Ali Sina University and Nanozino Co. for their financial and technical support during the course of this study.
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Figures
Fig. 1. Structures of scopolamine and atropine
Fig. 2. QDs-M2R/ Sc-Rho123 complex leads to the occurrence of the FRET system between the two fluorophores (Cd/Te QDs and Rho123).
Fig. 3. FT-IR spectra of a) scopolamine before oxidation (the region marked with a blue star indicates the OH group of scopolamine), b) scopolamine after oxidation, the peaks marked with orange stars show the C=O bond at 1731.98 cm-1 and the CHO at 2853.89 cm-1 both indicating successful oxidation of scopolamine.
Fig. 4. The peak at 1650.56 cm-1, shown with a red star, corresponds to the N=C bond and the imine formation.
Fig. 5. A: Absorption and emission wavelengths of the pure solution of the Cd/Te QDs and Rho123. 1) Absorption peak of the pure Cd/Te QDs. 2) Emission wavelength of the pure Cd/Te QDs. 3) Emission wavelength of the Rho123. B: TEM image and DLS analysis of the TGA-capped Cd/Te QDs.
780 bp
Fig. 6. PCR analysis for the validation of A. rhizogenesis- mediated gene transfer to A. belladonna: 1- DNA ladder, 2- positive control (plasmid DNA from the AR15834 strain). 3, 4, 5 and 6 are related to the hairy roots, leaves, and stems of the transgenic plants, respectively. 7- Non transgenic root of A. belladonna.
Fig. 7. Optimization of the nanobiosensor, the graph is related to the QDs-M2R and the Sc-Rho123 conjugates. The red arrow shows the excitation and emission wavelengths of the QDs-M2R conjugate as donor (350 and 430 nm, respectively), and the blue arrow indicates the emission wavelength (568 nm) of the Sc-Rhod123 conjugate as acceptor.
Fig. 8. Calibration curves for determination of scopolamine hydrochloride. The response of the nanobiosensor is dependent on the concentration of scopolamine hydrochloride and the addition of various concentrations of scopolamine hydrochloride led to the fluorescence quenching of Rho123.
Fig. 9. The obtained spectra of the sensor exhibited remarkable scopolamine-dependent changes in extracts obtained from (a) hairy root, (b) stem, (c) leave, and (d) root of Datura anoxia as positive control, while no changes were observed in root extract of G. glabra as negative control (e).
Table 1 Comparison between the linearity range (LR), the limit of detection (LOD) and relative standard deviation (RSD) of the systems currently used for the detection of scopolamine and the nanobiosensor developed in the present work. Detection method
LR
LOD
0.8 - 80 µg.mL
-1
1.87 - 104 ng.mL HPLC
RSD (%)
Ref
1.92 μg.mL
4.1-10.7
[29]
0.8 ng
2
[34]
ND
[35]
5.0
[36]
5.4
[37]
ND
[38]
-1
-1
8- 200 μg.mL
-1
0.8
0.5–500 μg.mL
0.5 μg.mL
-1
100-10,000 ng.mL
-1
1 μg.mL
-1
-1
10 - 10,000 ng.mL
–1
–1
0.10 ng.mL
LC-MS GC-MS
10.38 - 1038 μg.mL-1 2-100 pg. mg-1 2-100 pg.mg-1
5.17 μg.mL-1
potentiometric sensor
10-2 - 10–6 mol.dm–3
8×10-7 mol·dm–3
capillary electrophoresis
50–2,000 μ mol.L
5 μmol.L
-1
2 pg.mg
-1
0.636 2 pg mg-1 ND
-1
1.5 1.6
50 μmol.L
4.3
0.001 μmol.L-1
1.62
-1
FRET using QDs
0.01-4 μmol.L-1
[39] Not reported [17] [15] [40]
Present work
[17
Highlights
A novel, sensitive, selective and rapid optical nanobiosensor method is developed This method based on FRET, nanobio conjugation of CdTe-TGA-M2 muscarinic receptor and conjugation of scopolamine- rhodamine123 as donor / acceptor pair, respectively. This system, responses for scopolamine hydrochloride (Tropane alkaloids) determination in extract of hairy roots of Atropa belladonna. Nanobiosensor be able easily for in-vitro determination of anticholinergic agent in plant extracts.
PII: DOI: Reference:
S0039-9140(16)30954-7 http://dx.doi.org/10.1016/j.talanta.2016.12.013 TAL17103
To appear in: Talanta Received date: 27 May 2016 Revised date: 2 December 2016 Accepted date: 6 December 2016 Cite this article as: Fereshte Bagheri, Khosro Piri, Afshine Mohsenifar and Smaiil Ghaderi, FRET-based nanobiosensor for detection of scopolamine in hairy root extraction of Atropa belladonna, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.12.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
FRET-based nanobiosensor for detection of scopolamine in hairy root extraction of Atropa belladonna Fereshte Bagheri 1, 21, Khosro Piri 1,*2, Afshine Mohsenifar 33, Smaiil Ghaderi 4 1
2
Department of Biotechnology, College of Agriculture, Bu-Ali Sina University, Hamedan, Iran Research pharmaceutical center, School of pharmacy, Kermanshah University of Medical Sciences,
Kermanshah, Iran 3
4
Research and Development Department of Nanozino, Tehran, Iran Department of Chemistry. Bu-Ali Sina University, Hamedan, Iran
[email protected] [email protected] [email protected] * Author to whom correspondence should be addressed; Tel.: +98 918-813-0783; Fax: +9821-82233470.
External Editor:
1
Speciality: Biosensor- Biotechnology- Drug Delivery Systems, Academic Status: Researcher, MS: Biotechnology, BS/BA: Agriculture engineering, Adress: Research pharmaceutical center, School of pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran 2 Speciality: Biotechnology, Academic status: Assistant professor 3 Speciality: Biosensors - Drug Delivery Systems, Academic Status: Assistant Professor, PhD: Clinical Biochemistry , MS: Clinical Biochemistry , BS/BA: Laboratory Sciences, Address: Research & Development Department of Nanozino, Tehran, Iran. Phone: 021-77061120; Fax: 021-44787371
ABSTRACT A simple, sensitive, selective, and rapid optical nanobiosensor based on FRET was designed to detect tropane alkaloids as anti-cholinergic agents in natural and transgenic hairy roots extracts of Atropa belladonna. To achieve that, conjugation of tioglycolyic acid capped cadmium telluride quantum Dots, M2 muscarinic receptor (Cd/Te QDs-M2R) and conjugation of scopolamine-rhodamine123 (Sc-Rho123) were performed. More specifically, proportional amounts of M2 muscarinic receptor and quantum dots (QDs) were conjugated while scopolamine (as a tropane alkaloid) and rhodamine123 were also combined and these moieties functioned as donor and acceptor pairs, respectively. The system response was linear over the range of 0.01-4 µmol.L-1 of scopolamine hydrochloride concentration with a detection limit of 0.001 µmol.L-1. The developed nanobiosensor was successfully used for in vitro recognition of scopolamine as an anti-cholinergic agent in the investigated plant extracts. In addition, Agrobacterium rhizogenesis mediated gene transfer technique was employed to generate hairy roots and to enhance the production of tropane alkaloids in the studied medicinal plant.
Graphical Abstract
Construction of CdTeTGA-M2-ScRhod complex by combination of CdTe-TGA-M2, ScRhod conjugation and FRET system between two fluorophores, CdTe and rhodamine123 established.
Keywords: Nanobiosensor; FRET; Cd/Te Quantum Dots; M2 muscarinic receptor; Tropane alkaloids; Hairy roots
1. Introduction Nanoparticles are of considerable interest in the biotechnological field owing to their wide applications in drug delivery, in vitro and in vivo monitoring in cellular research, nucleic acid analysis, and signal transduction. Moreover, their unique electronic and magnetic properties have marked them as excellent materials for designing and developing sensors and biosensors [1]. Among various types of nanobiosensors, FRET-based ones due to their rapid and highly sensitive mechanisms have attracted a lot of attention for quantification of different target analyts. In fact, FRET-based nanobiosensors are considered as powerful instruments offering specific applications, e.g., in vitro and in vivo sensing of protein- protein interactions, receptor/ligand interactions, medicinal diagnostics, estimating of DNA/RNA concentration as well as in cancer research. It is worth quoting that FRET-based
nanobiosensors have greatly simplified experimental measurements owing to the fact that their quantitative assays are easily performed [2- 5]. The emergence of semiconductor quantum dots (QDs) and their combination with FRET have greatly enhanced the application of this technology within the field of biotechnology. In fact, recent years have witnessed a fast growth in the number and diversity of QDs nanoparticles, FRET-based biosensors developed [4]. QDs nanoparticles are fluorescent materials which illustrate excellent photo-physical properties such as high brightness and photostability. Among the II-IV semiconductors, Cd/Te QDs nanoparticles are considered as promising material for various biological applications owing to their attractive features, i.e., broad excitation spectrum, narrow emission spectrum, fluorescent quantum yields, facile synthetic versatility and having a direct band gap as well as bulk band gap energy of 1.48 eV which are suitable for sensors designing. Hence, the uses of Cd/Te QDs have been particularly advantageous in the field of biosensors development [5]. Various stabilizers have also been used in the synthesis of QDs [4- 6]. Among which, many sensors have been established based on the surface interaction of thioglycolic acid and a specific detecting moiety. Therefore, functionalized QDs have been introduced as one of the most important fluorescent sensors for many analyts. In fact, expanding the application of the capped QDs to develop fluorescent sensors in aqueous media is a topic of current interest. Earlier studies related to the interactions among QDs and many analyts have revealed that surface capping ligands have noticeable effects on the fluorescence response of QDs to physiologically
important
biomolecules
such
as
ions,
small
molecules
and
biomacromolecules [6]. Anticholinergics are a class of drugs used for treating diseases like asthma, incontinence, gastrointestinal cramps, and muscular spasms [2]. They are also prescribed for depression and sleep disorders [3]. Their action mechanism involves the blocking of the
neurotransmitter acetylcholine in the brain [7]. Tropane alkaloids as anticholinergic and pharmaceutical agents can competitively bind to the neurotransmitter, acetylcholine, in the central nervous system. Their targets are either muscarinic acetylcholine receptors or, less commonly, nicotinic acetylcholine receptors [8]. Atropa belladonna plant is a very rich source of tropane alkaloids such as scopolamine, hyocsyiamine and its racemic form atropine (Fig.1) [9]. It is well-documented that genetic modifications can increase the production of metabolites of interest. In fact, rapid growth of root cultures and significant increase of many secondary metabolites can be established either by manipulation of auxin levels in the culture medium or by genetic transformation of plants with Agrobacterium rhizogenesis [10-11]. A. rhizogenes is able to transform plant cells by introducing a part of its Ri-plasmid (T-DNA) into the plant genome [12-13]. Pharmaceutical applications of tropane alkaloids have led to an interest for precise determination of these active substances in medicinal plants. Various analytical methods such as TLC, HPLC, GC, GC/MS and LC-MS have been reportedly employed for the detection and quantification of tropane alkaloids in medicinal plants [14]. Furthermore, detection of tropane alkaloids especially scopolamine and atropine in pharmaceutical formulations have also accomplished using potentiometric sensor [15], atropine sulfate bulk acoustic wave sensor [16], LC-MS-MS [17], electro-chemiluminescance electrospun carbon nanofiber based sensor [18], as well as β-cyclodextrin modified ion sensitive field effect transistor sensor [19]. However, all these methods are expensive and time-consuming. On the contrary, nanobiosensor systems are novel, sensitive, selective, fast, reliable, and simple. More specifically and as mentioned earlier, FRET-based nanobiosensors have been known for their superior sensitivity and applicability for negligible volumes of specific pharmaceuticals [20]. Nevertheless, to the best of our knowledge, the use of functionalized Cd/Te QDs as Förster resonance sensor for selective recognition of anticholinergic drugs is
virtually unexplored. This is the first report on quantification analysis of tropane alkaloids in natural and hairy root extracts by using a FRET-based nanobiosensor (Fig. 2). To achieve that, M2 muscarinic receptor was applied as a biorecognition element. It is worth noting that M2 muscarinic receptor (M2R) belonging to G protein-coupled receptors (GPCRs) has substantial roles in both central and parasympathetic nervous systems. This receptor acts as an essential protein for physiological control of cardiovascular function [21]. TGA-capped Cd/Te QDs nanoparticles were synthesized and conjugated to M2 muscarinic receptor as donor. On the other hand, scopolamine-Rho123 conjugation was prepared as acceptor. The synthesized conjugates were then used in a FRET-based nanobiosensor system for the detection of tropane alkaloids in the natural extract and the hairy root extract of A. belladonna transformed by A. rhizogenesis, AR15834 strain. Suggested place for Fig. 1
Suggested place for Fig. 2 2. Experimental 2.1. Reagents Seeds of A. belladonna were collected from a medicinal plants garden located in Hamadan, Iran. A. rhizogenesis, AR15834 strain, was purchased from the Biotechnology Research Center, Karaj, Iran. Scopolamine hydrochloride, Rho123, CdCl2.2.5H2O, NaBH4, Tellurium
powder,
(TGA),
tetramethylammonium
hydroxide,
1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma (Germany). M2 muscarinic receptor was obtained from Jackson Immuno research Laboratory (USA). All the other chemicals used were purchased from Merck (Germany). 2.2. Experimental apparatuses
DNA amplification was performed by using a Bio-Rad thermo cycler (USA). FT-IR analysis was performed on a Perkin Elmer FT-IR spectrum (USA). The transmission electron microscopy (TEM) image was obtained by using a Philips H600 (Netherland). All fluorescence spectra were recorded by Perkin-Elmer LS-50B fluorescence Spectrometer (USA).
2. 3. Oxidation of primary alcohol of scopolamine In order to attach scopolamine to Rho123, the primary alcohol of scopolamine oxidized to the aldehyde. Barium manganate (BaMnO4), an oxidizing agent can use as a very effective catalyst for selective oxidation of primary alcohols to aldehydes [22]. Briefly, 102 mg of scopolamine was dissolved into 40 ml of chloroform and then 1.5 g of BaMnO4 was added to the mixture. The reaction mixture was kept under magnetic stirring and reflux for 20 min. Reaction progression was monitored by a TLC in petroleum ether/chloroform (50:50 v/v). Subsequently, the mixture was dried over anhydrous Na2SO4, and filtered with chloroform. Finally, the solvent was evaporated and the dried product was purified by a TLC plate in petroleum ether/chloroform (50:50 v/v).
2.4. Conjugation of scopolamine to Rho123 After scopolamine was oxidized to its corresponding aldehyde, it was conjugated to Rho123 through the primary amine of the dye as described previously by Reddy Chereddy [23] with some modifications. In brief, under reflux and N2 bubbling condition, 10 mg of Rho123 was dissolved in 30 ml of methanol, and then 10 mg the oxidized scopolamine was added slowly. The mixture was agitated at 60°C for 6 h to yield a dark purple solution. Furthermore, the progress of reaction was monitored by TLC. Then, the mixture was cooled at room temperature. Subsequently, it was filtered and washed 3 times with methanol. The
resulting solution was dried at room temperature. Finally, the FT-IR spectrum of the generated product was taken to prove imine bound formation.
2.5. Preparation of TGA-capped Cd/Te QDs Colloidal TGA-capped Cd/Te QDs were prepared according to the literature [24- 25]. In brief, 100 mg of Te powder was reduced by 280 mg of NaBH4 in 7 ml of double-distilled water under N2 atmosphere and magnetic stirring. After 5 h, the color of the obtained solution (NaHTe) was changed from violet to white. On the other hand, 358 mg CdCl2.2.5 H2O and 0.2 ml of TGA were dissolved in 200 mL deionized water and the pH of the solution was adjusted to 10 by drop-wise addition of NaOH solution (1 M). The produced Cd2+ solution was then de-aerated under nitrogen bubbling. Subsequently, the H2Te gas was obtained by reacting the NaHTe solution with diluted H2SO4 (0.5 mol.L-1) and was then passed through the oxygen-free Cd2+ solution with a gentle nitrogen flow (under stirring condition). The mixture was refluxed under nitrogen atmosphere together with sever stirring. By prolonging the refluxing time, different sizes of QDs was achieved. In order to eliminate excess precursors, the crude solution was washed three times with ethanol and centrifuged at 10,000×g for 15 min. Then, the precipitate was re-dispersed in 250 mL of water and kept at 4°C in darkness. Maximum excitation and emission wavelengths were 375 and 505 nm, respectively.
2.6. Conjugation of TGA-capped Cd/Te QDs and M2 muscarinic receptor Since the synthesis of the Cd/Te QDs was performed in an aqueous solution and owing to carboxylic group of the capping agent, the surface of the QDs was negatively charged. For conjugating the QDs to M2 muscarinic receptor, firstly, 1ml of the prepared QDs solution was mixed in to 5 ml of EDC (1 mg) and NHS (1 mg) solution at pH 6 and was
incubated in darkness for 1h at room temperature to ensure the attachment of EDC and NHS onto the surface of the QDs. Then, appropriate mounts of an ice-chilled solution of M2 muscarinic receptor was added to the activated QDs solution under mild magnetic stirring and this condition was maintained for 1h. Therefore, positively charged M2 muscarinic receptor interacted with the QDs through charge transfer mechanism. The nanobioconjugate stock solution was stored at 4°C in darkness until use.
2.7. Construction of the QDs-M2R/Sc-Rho123 To verify the formation of QDs-M2R/ScR- Rho123 complex, 2 ml of phosphate buffer (pH 7, 50 mM) and 0.5 mL of the QDs-M2R complex solution (as donor) were initially introduced into a fluorimeter cell and subsequently 0.5 mL of the Sc-Rho123 complex solution (as acceptor) was added to the reaction mixture. After incubation for 3 min, successful construction of the QDs-M2R/Sc-Rho123 complex was investigated through spectrofluorometry (excitation at 350 nm; 25 nm bandwidth for both excitation and emission spectra). 2.8. Hairy root induction Seeds of A. belladonna were cultured on 1/2MS medium. Then, the pieces of leaves, stems, and roots of 14-28 days plantlets were infected by A. rhizogenesis, AR15834 strain, containing Ri-plasmid. The plantlets inoculated with the Agrobacterium were cultured in 1/2MS medium containing cefotaxime (500 mg.L-1). After 7-10 days, hairy roots of A. belladonna were observed. The hairy roots were sub-cultured in 2 weeks intervals. Regeneration of transgenic plants from hairy roots can be either spontaneous or can be induced with the help of plant growth regulators. In the present study, spontaneous plantlets regeneration was observed in hairy root cultures after 3 sub-culturing passages. Plantlets regeneration was optimized in a greenhouse as elaborated [26]. Hairy roots, leaves, and stems
of the regenerated plants were extracted using the method described by Kamada et al. [27]. All samples were kept at 4°C and were used for detection of tropane alkaloids by using the designed nanobiosensor system. 2.9. DNA analysis To ensure the formation of transgenic hairy roots and the regenerated plantlet, DNA was extracted using the CTAB method [28]. PCR analysis was performed using specific primers to confirm the presence of the rol B gene in the hairy root, leaves, and stems of the regenerated plantlets. Natural roots and the plasmid A. rhizogenesis were used as negative and positive controls, respectively.
2.10. Evaluation of nanobiosensor for assessment of tropane alkaloids in plant extracts Following the optimization of the developed nanobiosensor and for its assessment, a solution including 2 ml of phosphate buffer (50 mM, pH 7), 50 μl of the QDs-M2R nanobioconjugate, and 50 μl of the Sc-Rho123 conjugate was added to the detection system. Subsequently, different concentrations of the plant extracts including those of hairy roots, leaves, stems, and the root extract of Datura innoxia (as positive control) and Glycyrrhiza glabra (as negative control) were used for assaying their tropane alkaloids contents (Fig. 7). 3. Result and discussions Tropane alkaloids (scopolamine and hyoscyamine) in hairy roots, plants and other environmental and biological samples have been analyzed by routinely analytical methods such as HPLC [29- 30], GC [31], GC/MS [32- 33], and liquid chromatography-mass spectrometry [34] which as mentioned earlier are expensive and time-consuming. A review of some analytical methods used for quantification of scopolamine in biological samples is presented in Table 1.
Suggested place for Table 1
FRET-based approaches are particularly applied tools for sensing, monitoring, and discovering biological compounds. These systems have been successfully used previously for various applications, e.g., detection of porcine reproductive and respiratory syndrome [41], determination of dopamine [42], detection of bovine serum albumin (BSA) in micelles [43]. Kattke and et al. also applied a similar system for determination of Aspergillus amstelodami [44] while Shanehsaz and et.al developed a FRET-based nanobiosensor for identification of Helicobacter pylori [24]. Here, we report on a novel, sensitive, and simple optical nanobiosensor based on FRET using M2 muscarinic receptor and Cd/Te QDs for quantification of tropane alkaloids in plant extracts.
3.1. Oxidation of scopolamine To construct the nanobiosensor, the primary alcohol scopolamine was oxidized using barium manganate to its corresponding aldehyde. FT-IR spectrum of the purified product was performed for verifying the oxidation of scopolamine to the corresponding aldehyde. The IR spectrum revealed the presence of C=O stretching vibrations at 1731.98 cm-1 and CHO flexural at 2853.89 cm-1 confirming the elimination of OH from scopolamine (Fig. 3). Suggested place for Fig.3
3.2. Sc-Rho123 conjugate formation For preparing the Sc-Rho123 conjugate, the aldehyde group of the scopolamine was bound to the amine agent of rhodamine123, and the imine formation was proved by the FTIR spectrum obtained at the wave length range of 400- 4000 cm-1 (Fig. 4). As displayed, the
intensity of the band at 1650.56 cm-1 was correspond to the imine bond (N=C) indicating the successful attachment of scopolamine to Rho123. Suggested place for Fig. 4
3.3. Characterization of the Cd/Te QDs
The Cd/Te QDs due to their highly wide emission spectrum of high resolution can be applied in sensing many specific quenching materials. Moreover, modification of the outer surface of QDs with anionic carboxylate groups is one of the most commonly applied methods for dispersing QDs in an aqueous solution [4-5]. To achieve that, TGA was used in the present study, followed by the activation of the carboxylic groups of the TGA-capped Cd/Te QDs by EDC/NHS. Absorption and emission wavelengths of the prepared Cd/Te QDs and Rho123 solutions are displayed in Fig. 5. As demonstrated, the results obtained indicated that the maximum emission wavelength of the QDs was at 505 nm, while the maximum absorption and emission wavelengths of Rho123 were at 510 nm and 580 nm, respectively. The full width at half maximum (FWHM) of the QDs emission wavelength was at about 35 nm. Emission spectra of the QDs and absorption wavelength of Rho123 showed maximum spectral overlap which is critical for gaining optimum FRET mechanism. As shown in Fig. 5, the TEM image and DLS analysis of the prepared TGA-capped Cd/Te QDs revealed particles of spherical shape with a particle size of about 3 nm and a good monodispersity. All these attributes indicated the suitability of the QDs for designing the nanobiosensor. At adequately basic pH, the electrostatic repulsion between the QDs maintained a colloidal suspension and a high optical stability without considerable reduction of fluorescence intensity during three months.
Suggested place for Fig. 5
3.4. Cd/Te QDs-M2R nanobioconjugate formation To ensure successful conjugation of the QDs and M2 receptor, the optical density (at 280 nm) of the sample containing the CdTe-M2R before and after centrifugation at 10,000 × g for 20 min at 4°C was recorded by a spectrophotometer. More specifically, the receptor had a typical peak at 280 nm in the absorption wavelength; whose absorbance intensity was reduced after the conjugation between the QDs and M2 receptor took place. This might be attributed to the chemical attachment of the M2 receptor onto the surface of the QDs through the EDC/NHS amidization protocol. In better words, this indicated that the covalent conjugation of the QDs and the M2 receptor was achieved [45- 47].
3.5. Transformation of leaves, stems, and root disks of A. belladonna by A. rhizogenesis and regeneration of hairy root The hairy root was induced in leaves, stems, and root disks of A. belladonna by A. rhizogenesis AR15834 strain. Many altered phenotypic characters, the so-called ‘‘Hairy Root Syndrome’’ can be created due to the combined expression of rol A, rol B, and rol C genes [11]. Rol B gene is associated with reduction in the length of stamens, protruding stigmas, and increase in adventitious roots on stems [10]. A large number of hairy roots were regenerated spontaneously within 6-8 weeks [12-13, 45]. Successful amplification of a 780 bp segment in the present study proved that the roots, leaves, and stems of the regenerated plants were successfully transformed and that they contained rol B oncogene while no amplification was observed for the roots, leaves, and stems of natural plants (Fig. 6).
Suggested place for Fig.6
3.6. FRET assessment FRET is a distance-dependent interaction between the electronic excited states of two dye molecules in which the excitation energy is transferred from a donor molecule to an acceptor molecule without emission of a photon. In fact, it is an important technique for investigating a variety of biological phenomena capable of making changes in molecular proximity. As mentioned above, the QDs-M2R/Sc-Rho123 complex was constructed by mixing of the QDs-M2R and Sc-Rho123 conjugates leading to the occurrence of the FRET phenomenon between the two fluorophores. Fig. 2 shows the occurrence of the FRET system between the two fluorophores (Cd/Te QDs and Rho123). 3.7. Nanobiosensor optimization The optimum excitation and emission spectra of the QDs-M2R conjugate as donor in the FRET system was identified by using its various concentrations The results obtained revealed that the concentration of the QDs-M2R conjugate (150 μmol.L-1) led to the optimum excitation and emission wavelengths of 350 nm and 430 nm, respectively. Moreover, in order to determine the optimal condition of the designed system, the responses of the nanobiosensor to the different amounts of the Sc-Rho123 conjugate (50-300 μmol.L-1) as acceptor was also investigated. At the 150 μmol.L-1 concentration of the QDs-M2R conjugate, the optimum concentration of the Sc-Rho123 conjugate was found at 50 μmol.L-1 leading to the maximum emission spectrum of Rho123 at 568 nm (Fig. 7) In fact, the occurrence of the energy transfer (FRET) between the QDs and the Rho123 in the QDs-M2R/Sc-Rho123 complex was confirmed by measuring the fluorescence intensity in a spectrofluorimeter (Fig. 7). Upon the formation of the QDs-M2R/Sc-Rho123 complex, the FRET phenomenon took place due to the reasonable wavelength overlapping
between the donor and acceptor moieties. It should be noted that QDs have an extended range of absorption wavelengths. Therefore, a vast range of excitation wavelengths was selected. Nevertheless, the excitation wavelength of 350 nm was found suitable in the present study. Suggested place for Fig. 7
3.8. Standard curve Standard curve was plotted for scopolamine hydrochloride by competitively replacing scopolamine hydrochloride at variable concentrations (0.01-4 μmol.L-1) with Sc-Rho123 conjugate at the constant concentration of 50 μmol.L-1 (Fig. 8). As shown, a significant response to scopolamine hydrochloride increasing concentration was observed. More specifically, by increasing scopolamine hydrochloride concentration the fluorescence quenching of Rho123 was intensified. This revealed the accuracy of the constructed nanobiosensor in recognition of scopolamine. The correlation coefficient was recorded at 0.98, with a limit of detection (LOD) of 0.001 µmol.L-1, which was calculated based on the LOD=3S0.K-1 equation, where S0 is the standard deviation of the blank (n=4) and K is the slope of the calibration curve. Suggested place for Fig. 8
3.9. Recognition and measurement of scopolamine in plant extracts Determination of scopolamine in plant extracts was achieved by introduction of various concentrations of the extracts obtained from the hairy root, leaves and stem of A. belladonna and root of D. innuxia to the nanobiosensor (Fig. 9, parts a, b, c, d). Considering the data presented in these graphs, by adding an adequate values of the D. innuxia root extract, the emission peak of Rho123 was decreased. On the contrary, by adding G. glabra extract no changes were observed in the emission peak Rho123 (Fig. 9, part e). In fact, this
was indicative of the specificity and selectivity of the developed nanobiosensor. Finally, scopolamine was quantified in the hairy roots, leaves, and stems of A. belladonna and root of D. innuxia extracts at 0.081, 0.021, 0.001 and 0.01 mg.g-1, respectively. The amount of scopolamine in the hairy roots of A. belladonna, determined by HPLC in our previous report stood at 0.06 mg.g-1 [10-12].
Suggested place for Fig. 9
4. Conclusions A simple optical nanobiosensor based on FRET using M2 muscarinic receptor and Cd/Te ODs was developed herein for the identification/quantification of scopolamine in biological systems. As compared to the other methods reported for the detection and quantification of tropane alkaloids, low detection limit, high sensitivity and selectivity, fast response, and simplicity were readily achieved in the present work. Moreover, the shelf-life and the detection limit of the nanobiosensor were found 6 month and 0.001 µmol. L-1, respectively. The present nanobiosensor can be used in a wide range of fields such as plant metabolites, medicinal research, biomedical diagnostics, and drug discovery. Moreover, it should be highlighted that muscarinic receptors are unique targets for treatment of disease such as Parkinson, Schizophrenia, Alzheimer, chronic obstructive pulmonary diseases [21]. Therefore, the designed nanobiosensor through selective assessment of orthosteric drugs can also assist in treatment of these disorders.
Acknowledgments The authors are grateful to Bu-Ali Sina University and Nanozino Co. for their financial and technical support during the course of this study.
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Figures
Fig. 1. Structures of scopolamine and atropine
Fig. 2. QDs-M2R/ Sc-Rho123 complex leads to the occurrence of the FRET system between the two fluorophores (Cd/Te QDs and Rho123).
Fig. 3. FT-IR spectra of a) scopolamine before oxidation (the region marked with a blue star indicates the OH group of scopolamine), b) scopolamine after oxidation, the peaks marked with orange stars show the C=O bond at 1731.98 cm-1 and the CHO at 2853.89 cm-1 both indicating successful oxidation of scopolamine.
Fig. 4. The peak at 1650.56 cm-1, shown with a red star, corresponds to the N=C bond and the imine formation.
Fig. 5. A: Absorption and emission wavelengths of the pure solution of the Cd/Te QDs and Rho123. 1) Absorption peak of the pure Cd/Te QDs. 2) Emission wavelength of the pure Cd/Te QDs. 3) Emission wavelength of the Rho123. B: TEM image and DLS analysis of the TGA-capped Cd/Te QDs.
780 bp
Fig. 6. PCR analysis for the validation of A. rhizogenesis- mediated gene transfer to A. belladonna: 1- DNA ladder, 2- positive control (plasmid DNA from the AR15834 strain). 3, 4, 5 and 6 are related to the hairy roots, leaves, and stems of the transgenic plants, respectively. 7- Non transgenic root of A. belladonna.
Fig. 7. Optimization of the nanobiosensor, the graph is related to the QDs-M2R and the Sc-Rho123 conjugates. The red arrow shows the excitation and emission wavelengths of the QDs-M2R conjugate as donor (350 and 430 nm, respectively), and the blue arrow indicates the emission wavelength (568 nm) of the Sc-Rhod123 conjugate as acceptor.
Fig. 8. Calibration curves for determination of scopolamine hydrochloride. The response of the nanobiosensor is dependent on the concentration of scopolamine hydrochloride and the addition of various concentrations of scopolamine hydrochloride led to the fluorescence quenching of Rho123.
Fig. 9. The obtained spectra of the sensor exhibited remarkable scopolamine-dependent changes in extracts obtained from (a) hairy root, (b) stem, (c) leave, and (d) root of Datura anoxia as positive control, while no changes were observed in root extract of G. glabra as negative control (e).
Table 1 Comparison between the linearity range (LR), the limit of detection (LOD) and relative standard deviation (RSD) of the systems currently used for the detection of scopolamine and the nanobiosensor developed in the present work. Detection method
LR
LOD
0.8 - 80 µg.mL
-1
1.87 - 104 ng.mL HPLC
RSD (%)
Ref
1.92 μg.mL
4.1-10.7
[29]
0.8 ng
2
[34]
ND
[35]
5.0
[36]
5.4
[37]
ND
[38]
-1
-1
8- 200 μg.mL
-1
0.8
0.5–500 μg.mL
0.5 μg.mL
-1
100-10,000 ng.mL
-1
1 μg.mL
-1
-1
10 - 10,000 ng.mL
–1
–1
0.10 ng.mL
LC-MS GC-MS
10.38 - 1038 μg.mL-1 2-100 pg. mg-1 2-100 pg.mg-1
5.17 μg.mL-1
potentiometric sensor
10-2 - 10–6 mol.dm–3
8×10-7 mol·dm–3
capillary electrophoresis
50–2,000 μ mol.L
5 μmol.L
-1
2 pg.mg
-1
0.636 2 pg mg-1 ND
-1
1.5 1.6
50 μmol.L
4.3
0.001 μmol.L-1
1.62
-1
FRET using QDs
0.01-4 μmol.L-1
[39] Not reported [17] [15] [40]
Present work
[17
Highlights
A novel, sensitive, selective and rapid optical nanobiosensor method is developed This method based on FRET, nanobio conjugation of CdTe-TGA-M2 muscarinic receptor and conjugation of scopolamine- rhodamine123 as donor / acceptor pair, respectively. This system, responses for scopolamine hydrochloride (Tropane alkaloids) determination in extract of hairy roots of Atropa belladonna. Nanobiosensor be able easily for in-vitro determination of anticholinergic agent in plant extracts.