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Article Cite This: ACS Omega 2019, 4, 2948−2956
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Aptasensor for Quantifying Pancreatic Polypeptide Ahmed S. M. Ali,†,‡ Medhat S. El-Halawany,‡ Sherif Abdelaziz Ibrahim,‡ Olga Plückthun,§ Ahmed S. G. Khalil,*,† and Günter Mayer*,§,∥ †
Faculty of Science, Environmental and Smart Technology Group, Fayoum University, 63514 Fayoum, Egypt Faculty of Science, Department of Zoology, Cairo University, 12613 Giza, Egypt § Life and Medical Sciences Institute and ∥Center of Aptamer Research and Development, University of Bonn, 53121 Bonn, Germany ‡
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S Supporting Information *
ABSTRACT: Herein, a simple colorimetric aptasensor based on unmodified gold nanoparticles (AuNPs) has been developed for quantifying pancreatic polypeptide (PP), a peptide hormone associated with nonfunctional neuroendocrine pancreatic tumor (NF-PNET). First, several PP-specific aptamers have been screened by systematic evolution of ligands by exponential enrichment. The generated aptamers showed specificity and affinity higher than those reported so far. The sensing platform was constructed based on the optical properties of AuNPs as well as the adsorption of singlestranded DNA aptamers. The high sensitivity of the developed aptasensor was achieved by optimizing the sensing conditions, including salt tolerance, time of reaction, and optimal aptamer concentration. The developed aptasensor showed a wide linear dynamic range (1−60 nM) with a limit of detection of 521 pM. Furthermore, the performance of the aptasensor was evaluated for the detection of PP in human serum and showed recovery values ranging between 92.4 and 93.9%. Therefore, the presented colorimetric aptasensor has a great potential in the diagnosis of NF-pNETs.
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INTRODUCTION Pancreatic polypeptide (PP) is a 36 amino acids peptide hormone produced and secreted by PP cells (or F cells) located in the periphery and intermediate zone of pancreatic Langerhans islands.1,2 PP plays an important role in the reduction of energy demand by inhibition of stomach stimulation and pancreatic exocrine function in addition to the inhibition of hepatic glucose production and pancreatic insulin secretion.3 The basal PP level in serum is about 100 pM, and it depends on the age and gender of the person.4,5 An elevated level of PP in serum has been found in the association of a specific type of pancreatic cancer known as the nonfunctional neuroendocrine pancreatic tumor (NFPNET).4,6 Thus, the detection of an elevated level of PP will aid the early detection of NF-PNET. Recently, a radioimmunoassay (RIA) has been developed to quantify PP.7 Although RIA has shown high sensitivity and specificity for PP detection, it requires equipped radioactive laboratory and high handling precautions. This limitation was overcome by an enzyme-linked immunosorbent assay (ELISA) detecting PP,8 albeit ELISAs involve antibodies as molecular recognition elements, which have disadvantages, e.g., high production cost and short shelf half-life because of low stability at high temperatures and pH changes.9,10 These limitations call for approaches to substitute antibodies with other molecular recognition probes.11−15 Aptamers are short single-stranded © 2019 American Chemical Society
DNA (ssDNA) or RNA that bind to cognate target molecules with high specificity and affinity. They fold into specific threedimensional structures and are identified by a process termed systematic evolution of ligands by exponential enrichment (SELEX).16 Due to stability under different physiological conditions, cost-effectiveness, and easy modification, various aptamers have been developed against different target molecules and applied for disease diagnostics.17,18 The capability of aptamers to recognize a wide range of target molecules, including metal ions, amino acids, peptides, proteins, and whole cells, as well as nonimmunogenic targets like toxins make them ideal recognition elements in sensor applications.19−26 Over the last two decades, aptamers were used in many types of biosensors and proven to be compatible with various read-out formats.27−34 Colorimetric aptasensors based on gold nanoparticles (AuNPs) have been reported, especially for the detection of small molecules due to its easy preparation and simple operation.35,36 In general, this colorimetric aptasensor works in two different configurations. One is based on AuNPs modified with complementary oligonucleotides, which hybridize and capture aptamers.37 The other configuration is based on physical adsorption of Received: November 9, 2018 Accepted: January 29, 2019 Published: February 8, 2019 2948
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(R1, R3, R6, and R8) were selected for testing by flow cytometry. Therefore, a forward primer with a Cy5 residue at the 5′ end was used to generate fluorescently labeled aptamers by polymerase chain reaction (PCR), followed by exonuclease digestion. The thus prepared DNA molecules were incubated with PP-modified beads, and after incubation, the mean fluorescence intensity (MFI) was determined by flow cytometry. Values obtained of DNA from the enriched libraries were compared to the starting library (SL) and beads without ssDNA (Figure 2). Our results show a gradual
aptamers on the surface of unmodified AuNPs. The last configuration has been widely applied for the detection of many target molecules because of its easy and simple preparation steps.38−40 The protein corona is the main challenge facing applications of AuNPs-based biosensors in biological samples, as it influences the dispersion of AuNPs.41 To overcome this problem, many researchers have pretreated the real samples with denaturing agents to eliminate the high-molecular-weight proteins.42,43 Herein, a simple aptasensor for detection of PP based on unmodified gold nanoparticles has been developed. We have successfully identified specific aptamers binding to PP and investigated their dissociation constants (Kd) and selectivity. We also demonstrate the PP-dependent aggregation of AuNPs as a result of specific PP−aptamer binding. The conditions of biosensing, including salt tolerance, time of aggregation, and optimal aptamer concentration, have been optimized.
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RESULTS AND DISCUSSION Selection of PP-Aptamers. For the selection of aptamers binding to PP, we immobilized the target molecule on streptavidin magnetic beads. We also added (fetal calf serum (FCS)) in the incubation buffer. FCS contains a series of proteins that served as counter target molecules by which we sought to increase the competition for ssDNA sequences and drive the performance of the library toward PP-specific binders. A negative selection step has been included against the empty beads to minimize the enrichment of sequences toward streptavidin. We used lambda exonuclease for ssDNA displacement as it was reported to improve the final purity of the ssDNA solutions.44 Through the rounds of selection, stringency was increased gradually as shown in Table S2. A representative selection cycle is given in Figure 1. After eight selection cycles, the progression of the enrichment was monitored. DNA obtained from several selection rounds
Figure 2. Monitoring the progression of the selection targeting PP. Given is the mean fluorescence intensity (MFI) of different selection rounds (R1, R3, R6, and R8) and the starting library (SL). For each DNA from the indicated selection round, the obtained MFIs using PP coupled beads were compared to empty beads, monitoring the nonspecific binding of the enriched DNA libraries to streptavidin. The data show an increase of aptamer enrichment gradually from the first round to the eighth round. It also shows a decrease in nonspecific binding of aptamers to streptavidin in the third round compared to the first round. The error bar represents the standard deviation (SD) of two independent experiments, which were conducted in duplicate.
increase of aptamer enrichment from the third round to the eighth round. It also shows a slight decrease in nonspecific binding of aptamers to the streptavidin in the third round compared to the first one. This is most likely caused by the negative selection step applied from the third round on. Sequencing and Bioinformatic Analysis. The chromatogram files of sequenced colonies were analyzed using FinchTV software. The random regions were extracted between the forward primer and complement of the reverse primer. The data showed that the 47 sequenced colonies revealed 22 unique sequences, whereas the sequences S4 and S5 were most abundant (Table S3). The similarity distance was investigated by generating a phylogenetic tree for the 22 sequences using ClustalW.45 One sequence from each tree branch was selected according to the frequency number for further characterization (Figure S2). The sequences were also monitored for shared motifs with MEME suite.46 Two shared motifs with rich G content (Figure S3) were identified, which are indicative of G-quadruplex structure formation.47 This notion is further supported by G-score prediction, in which a score of 1 was obtained for all sequences (Table S4), as performed by QGRS.48 The secondary structures of the chosen eight sequences were additionally predicted by mfold,49 employing the selection conditions (25 °C, 1 mM Mg2+, and
Figure 1. Schematic representation of the applied aptamer selection process. The ssDNA library was incubated with the PP-modified beads. After incubation, the bound sequences were separated. To reduce the amount of matrix binders, a negative selection step using empty beads was applied. Enriched sequences were amplified, and the corresponding ssDNA was used in subsequent rounds. Eight selection rounds have been performed before sequencing of the enriched library. 2949
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137 mM Na+). The most stable structures with the lowest ΔG are shown in Figure S4. Evaluation of the Enriched Aptamers. We used flow cytometry to evaluate the binding properties of the selected sequences. Flow cytometry has been considered the best choice in our case since it provided the same conditions as in the selection process. The experiments were conducted using aptamers labeled with Cy5 (300 nM). Our results showed reasonable binding for all sequences. Aptamers S2, S12, and S20 showed more than 90% of binding, as shown in Figure 3A. Therefore, the three sequences were selected for further analysis. Flow cytometry was used to determine the Kd values of the selected sequences. To eliminate the basal fluorescence and nonspecific binding to the beads, empty beads were incubated with the tested aptamers in parallel to the immobilized PP for all given concentrations. The change of fluorescence (ΔF) was determined for all concentrations by subtracting the fluorescence of the empty beads from that of the immobilized PP. A scrambled sequence of aptamer S12 was used as a negative (nonbinding) control in addition to the starting library (SL) (Figure 3B). The obtained isotherms were fitted by the Hill slope equation (Y = Bmax × Xh/(Kdh + Xh)), where Bmax = maximum specific binding, Kd = dissociation constant, h = Hill slope, X = aptamer concentration (nM), and Y = fluorescence change. The three selected sequences showed Kd values in the two-digit nanomolar range, with S20 revealing the highest affinity and a Kd value of 13 nM. These values are better than the reported aptamers targeting PP, which have Kd values of 33.15, 58.81, and 77.39 nM.50 To analyze the specificity of the aptamers, human serum albumin (HSA) and hemoglobin (Hb) were used as controls due to their abundance in plasma. As shown in Figure 3C, the aptamers S2, S12, and S20 bind to PP but not to HSA and Hb at a concentration of 10 nM. All experiments were conducted twice in duplicate. Synthesis and Characterization of AuNPs. The synthesized AuNPs were characterized by UV−vis spectrophotometry and high-resolution transmission electron microscopy (HRTEM). As shown in Figure S5, the UV−vis spectra showed a sharp peak at 520 nm indicating an average particle size of 15 nm.51 The concentration of AuNPs solution was about 9.75 nM, as determined by the Beer−Lambert law taking into consideration the absorbance of 1.97 at 520 nm and the molar extinction coefficient of 2.01 × 108 M−1 cm−1 52 in Figure 4A,B. The analysis of HRTEM images showed monodisperse, crystalline, and homogenous AuNPs. The particles were aggregated once NaCl was introduced (Figure 4C,D). Colorimetric Detection of PP. It has been reported that ssDNA could easily be adsorbed on the surface of AuNPs due to the electrostatic interaction between the AuNPs and bases of ssDNA. This high adsorption is a result of the flexibility of ssDNA, so more bases are facing the surface of AuNPs, unlike dsDNA, which have relatively rigid structure.53 The strong adsorption makes AuNPs more stable by increasing the repulsion between negatively charged particles and prevents the aggregation at high ionic strength. The ssDNA aptamers detach the surface of AuNPs by increasing the concentration of aptamer target (PP) due to the difference in the binding affinities. By desorption of aptamers, the AuNPs start to aggregate at high ionic strength, leading to a shift in color from red to purple-blue.54 Prior to PP detection, we investigated the optimal sensing conditions, including salt tolerance (minimum
Figure 3. Evaluation of aptamers. (A) Percentage of binding for the selected eight PP-aptamers (S1, S2, S4, S5, S8, S10, S12, and S20) at 300 nM representing the eight branches of the phylogenetic tree. All aptamers were compared to the starting library (SL) and the DNA from the last selection round (R8). For each aptamer, the PP beads were compared to empty beads to monitor the nonspecific binding of aptamers to streptavidin. (B) Kd determination for S2, S12, and S20 aptamers compared to a scrambled S12 aptamer and the starting library (SL). The error bars represent the standard deviation of two independent experiments, which were conducted in duplicate. (C) Specificity test for S2, S12, and S20 using HSA and Hb as controls.
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Figure 4. TEM images of the synthesized AuNPs. (A) The asprepared AuNPs showed good monodispersity of average particle size of 15 nm. (B) High resolution for one nanoparticle showing the lattice planes (inset, diffraction pattern). (C, D) Aggregated AuNPs at low and high magnification; the aggregation is induced by adding NaCl.
salt concentration required for AuNPs aggregation), aggregation time (time required to get stable aggregated particles), and optimal aptamer concentration. For investigating the salt tolerance, AuNPs were added to different concentrations of NaCl (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mM). The change of color was observed, and 625/520 nm absorbance ratio was determined.54 As shown in Figure 5A, 25 mM NaCl could induce AuNPs aggregation; the degree of AuNPs aggregation increased until it became completely aggregated at 45 mM NaCl. On the basis of this finding, 45 mM was selected as the optimal NaCl concentration. Subsequently, the time of aggregation was determined by UV−vis spectroscopy. Therefore, kinetic absorbance scan (400−800 nm) was done for the AuNPs after adding the optimal salt concentration. The kinetic spectra showed an increase of 625/520 nm absorbance ratio until it became stable after 3−5 min, as shown in Figure 5B. The optimal aptamer concentration was determined by incubation of different concentrations of aptamer (S12) (0, 20, 40, 60, 80, 100, 120, 140, 160, and 180 nM) with AuNPs for 60 min at 25 °C, followed by exposing to the optimal salt concentration (45 mM). The change of color was observed and 625/520 nm absorbance ratio was determined. As shown in Figure 5C, 45 mM NaCl caused complete aggregation of AuNPs when the aptamer concentration was zero, while 40 nM of S12 caused the maximum protection against aggregation. Figures 6 and 7 showed the specific detection of PP by the developed sensing platform. The aggregation was qualitatively screened by changing of color from red to blue and quantitatively by absorbance ratio 625/520 nm, the color of aggregation increased gradually starting from 1 nM until it became almost saturated at 1 μM. The similar structure peptide (NPY) and BSA were examined at the same conditions, and NPY showed no
Figure 5. Optimal sensing conditions. (A) The absorbance ratio 625/ 520 nm of AuNPs under various concentrations of NaCl. The dashed line represents the saturated state of aggregation. (B) Kinetic absorbance spectra for aggregation of AuNPs with 45 mM NaCl; the stable aggregation was reached after 3−5 min. (C) The absorbance ratio 625/520 nm of AuNPs under various concentrations of aptamer S12 in the presence of 45 mM NaCl. The dashed line represents the minimum aptamer concentration required to protect 2951
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Figure 5. continued the AuNPs from aggregation. The error bars represent that the standard deviation of two independent experiments conducted in duplicate.
Figure 6. Colorimetric detection of PP. The absorbance ratio 625/ 520 nm of AuNPs under different concentrations of PP in the presence of 45 mM NaCl and 40 nM aptamer S12. The data showed an increase in the degree of aggregation starting from 1 nM until it became almost saturated at 1 μM. The dashed line represents the maximum degree of aggregation. The error bars represent the standard deviation of two independent experiments conducted in duplicate.
significant response with a concentration of less than 80 nM, while BSA did not show any response up to 5 μM. The limit of detection (LOD) for PP was determined by the following equation55
Figure 7. Specificity of the colorimetric aptasensor. The absorbance ratio 625/520 nm of AuNPs under different concentrations of (A) neuropeptide Y (NPY) and (B) bovine serum albumin (BSA) in the presence of 45 mM NaCl and 40 nM aptamer S12. The data showed no response for NPY less than a concentration of 80 nM, while BSA did not show any response even at high concentration. The error bars represent the standard deviation of two independent experiments conducted in duplicate.
LOD = 3.3σ /S
where σ is the standard deviation of intercept and S is the slope of the linear regression response. For our developed biosensor, LOD was about 521 pM, which is reasonable for diagnosis of NF-PNET. The performance of the developed aptasensor was further evaluated for the detection of PP in real samples by spiking different concentrations of PP in human serum and determining the recovery values. Prior to the recovery experiments, the serum samples were treated with acetonitrile to remove high-molecular-weight and high-abundance proteins by forming a buff precipitate.56 Furthermore, the supernatant was filtered by a centrifugal column with 10 kDa molecular weight cutoff to ensure complete removal of high-molecularweight proteins. As shown in Table 1 and Figure 8, the recovery values ranged from 92.4 to 93.95%, which indicates the reliability of the developed aptasensor.
Table 1. Recovery of PP from Human Serum Samples serum sample
added PP (nM)
found (nM)
recovery (%)
1 2 3
15 30 50
14.09 28.11 46.2
93.95 93.72 92.40
low Kd values. Our sensing platform exhibits a wide linear detection range with a low limit of detection (521 pM) and high recovery in human serum (92.4−93.9%). The change of color resulting from the formation of PP−aptamer complexes and aggregation of AuNPs could be easily detected by the naked eye as well as UV−vis absorption spectroscopy, thus allowing a simple detection of PP without any additional modification.
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CONCLUSIONS In summary, we have developed a simple and sensitive colorimetric aptasensor for pancreatic polypeptide (PP) based on adsorption of ssDNA aptamers on the surface of unmodified AuNPs. The specific PP-aptamers have been generated by SELEX and showed high specificity for PP and 2952
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min at 25 °C and 800 rpm. After incubation, the beads were washed three times for 2 min with DPBS to remove unbound PP molecules. The resultant beads were resuspended in 50 μL of DPBS and used freshly in each selection round. In the first SELEX round, 500 pmol ssDNA library was incubated with the immobilized PP in binding buffer (pH = 7.0) (DPBS with 1 mM MgCl2 and 1 mM CaCl2) with 10% fetal calf serum (FCS, Thermo Fisher Scientific). The time of incubation was adjusted to be 30 min at 25 °C with shaking at 800 rpm. The aptamers bound to PP were eluted by heat treatment in ddH2O at 85 °C for 3 min, and the eluted aptamers were amplified by polymerase chain reaction (PCR). The PCR was conducted in eight parallel tubes in 100 μL reaction volume containing 12.5 μL of the supernatant, 47.5 μL of master mix, 2.5 μL of Taq polymerase (50 U/μL), and 37.5 μL of nucleasefree water. PCR was performed with initial predenaturation at 95 °C for 2 min, followed by three steps of denaturation at 95 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 45 s, and a final extension step at 72 °C for 2 min. The PCR product was subjected to 4% agarose (Genaxxon Bioscience GmbH, Germany) gel electrophoresis. The singlestrand displacement of the purified PCR product was carried out by lambda exonuclease treatment, followed by purification of the ssDNA. The stringency was increased through the eight selection rounds, and negative selection against empty beads has been included in rounds 3, 6, 7, and 8 directly before PCR. Characterization of the Generated Aptamers. Plasmid cloning and Sanger sequencing were conducted to identify enriched PP-aptamers. The PCR product of the last selection round was performed as described above, but the last extension step was extended for 25 min to permit the addition of a 3′ overhang deoxyadenosine. Then, the PCR products (last SELEX round and nontemplate control) were transformed into competent Escherichia coli with pCR 2.1 vector (One Shot TOP10, Invitrogen). DNA extraction of positively transformed clones was performed (NucleoSpin plasmid DNA purification kit, Macherey-Nagel, Germany), and its quality was checked by agarose gel electrophoresis. A total of 47 colonies were sequenced by Sanger sequencing57 at GATC Biotech (Germany), and Sanger reads were analyzed using FinchTV software (Digital World Biology). The secondary structures of the screened aptamers were predicted by mfold (web server for nucleic acid folding and hybridization prediction49). The binding properties of the enriched aptamers were monitored by flow cytometry (FACS Conto II, BD Biosciences). Briefly, the biotinylated PP was immobilized on the beads and incubated with Cy5 fluorophore-labeled aptamers. The fluorescence intensity was measured with flow cytometry, and data were analyzed with FlowJo software. The Kd values of the selected aptamers were determined and compared to the Kd values of the starting library and a scrambled sequence (same nucleotide content of a selected aptamer with different nucleotide order). The specificity of the selected aptamers for PP was monitored using human serum albumin (HSA) and hemoglobin (Hb) (Sigma-Aldrich, Germany) as controls. The Kd values were calculated by GraphPad Prism 6 software (GraphPad software). Construction of PP-Aptasensor Based on AuNPs. AuNPs were prepared by citrate reduction of gold chloride.58 In brief, 10.5 mL of 1% trisodium citrate was added to 300 mL of boiling HAuCl4 (0.01%). The reaction mixture was stirred at boiling temperature for 30 min and then left to cool down at room temperature with stirring. The size of synthesized AuNPs
Figure 8. Calibration plot of different PP concentrations vs absorbance ratio (625/520 nm). (A) The curve shows a linear increase of absorbance ratio with increasing PP concentration. The slope and standard error of the intercept are used to determine the limit of detection of the developed aptasensor. (B) Determination of PP in human serum. The error bars represent the standard deviation of two independent experiments conducted in duplicate.
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EXPERIMENTAL SECTION Selection of the PP-Specific Aptamers. All oligonucleotides, including ssDNA library and primers, were synthesized by Ella Biotech GmbH (Germany). The library contained a central randomized sequence of 43 nucleotides flanked by two constant regions composed of 18 and 19 bases representing forward primer and complement sequence of reverse primer, respectively (Table S1). The ssDNA library was denaturized before SELEX by heating at 90 °C for 5 min, followed by gradually cooling down (decline rate, ∼4 °C/min) to reach 25 °C. The denaturation step was also applied for ssDNA libraries from the subsequent selection rounds. Furthermore, biotinylated pancreatic polypeptide (Bachem, Switzerland) was immobilized on magnetic beads (Dyna M-280 Streptavidin, Thermo Fisher Scientific) prior to SELEX rounds. First, the beads were vortexed vigorously for 5 s and then resuspended, thrice, in 1× DPBS (KCl (200 mg/L), KH2PO4 (200 mg/L), NaCl (8 g/L), and Na2HPO4·7H2O (2.16 g/L)). Then, the equilibrated beads were incubated with biotinylated PP for 30 2953
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Funding
was determined by a high-resolution transmission electron microscope (JEOL Solutions for Innovation). To determine the concentration and check the characteristic peak of synthesized AuNPs, UV−visible absorption spectrum of the AuNPs was obtained using an Agilent 60 UV−visible spectrophotometer (Agilent Technologies) with a 1 cm path length cuvette. Prior to building the biosensor, AuNPs were washed to get rid of excess citrate by centrifugation for 30 min at 12 500 rpm, the supernatant was discarded, and the precipitated pellets were dissolved in the original ddH2O volume. Afterward, a mixture of 50 μL of AuNPs (9.75 nM) with 10 μL of aptamer S12 (0.4 μM) and 10 μL of different (PP) concentrations incubated with gentle rotation for 60 min at 25 °C. Then, 30 μL of NaCl (150 mM) was added and the change of color and spectrum were determined by the naked eye and UV−vis spectrophotometer.59 The concentration of PP was plotted against 625/520 absorbance ratio. The homologous peptide (NPY) and BSA were also examined at the same conditions to test the specificity of the developed sensing platform. Detection of Pancreatic Polypeptide in Human Serum. To overcome the effect of protein corona on AuNPs, we treated the serum with acetonitrile to eliminate the high-molecular-weight proteins.42,50 In detail, the blood samples, obtained from healthy volunteers (after fasting for 8 h), were centrifuged at 5000 rpm for 30 min at room temperature. The collected serum was added to acetonitrile with ratio 1:0.6 for serum and acetonitrile, followed by fortification with PP for different final concentrations (0, 15, 30, 50, and 60 nM). After centrifugation for 10 min at 10 000 rpm, the supernatant was collected and filtrated through a centrifugal filter device (Amicon Ultra 10 K) and concentrations of PP were measured in diluted serum 1:10. Statistical Analysis. All analytical performances were conducted in duplicate and repeated for at least in two independent experiments. The variability of data was represented by error bars as an indicator for the standard deviation (SD).
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This work was partially funded by Erasmus mobility program and funds from the German Research Council (DFG, SFB1089 and MA 3442/7-1 to G.M.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS S. Haßel, S. Kü nne, M. Choukeife, and L. Eiden are acknowledged for expert technical support.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03131. List of sequences of the ssDNA library and primers; summary of the SELEX rounds; progress of selection process by gel electrophoresis; list of unique sequences screened by Sanger sequencing; phylogenetic tree of the unique sequences; the most shared motifs; the predicted G-score; secondary structures predicted by mfold; and UV−vis spectrum for the synthesized AuNPs (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (A.S.G.K.). *E-mail:
[email protected] (G.M.). ORCID
Ahmed S. G. Khalil: 0000-0003-1825-6752 Author Contributions
The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. 2954
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DOI: 10.1021/acsomega.8b03131 ACS Omega 2019, 4, 2948−2956
ACS Omega
Article
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DOI: 10.1021/acsomega.8b03131 ACS Omega 2019, 4, 2948−2956
