Article pubs.acs.org/ac
Affinity Binding-Guided Fluorescent Nanobiosensor for Acetylcholinesterase Inhibitors via Distance Modulation between the Fluorophore and Metallic Nanoparticle Yaodong Zhang,*,†,‡ Tingting Hei,†,‡ Yanan Cai,†,‡ Qunqun Gao,†,‡ and Qi Zhang†,‡ †
Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, ‡Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China S Supporting Information *
ABSTRACT: The magnitude of fluorescence enhancement was found to depend strongly on the distance between fluorophores and metal nanostructures in metal-enhanced fluorescence (MEF). However, the precise placement of the particle in front of the molecule with nanometer accuracy and distance control is a great challenge. We describe a method using acetylcholinesterase (AChE) to modulate the distance between a gold nanoparticle (AuNP) and the fluorophore 7hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). We found that DDAO is a reversible mixed type-I AChE inhibitor. DDAO binds to the peripheral anionic site and penetrates into the active gorge site of AChE via inhibition kinetics test and molecular docking study. The affinity ligand DDAO bound to AChE which was immobilized onto AuNPs, and its fluorescence was sharply enhanced due to MEF. The fluorescence was reduced by distance variations between the AuNP and DDAO, which resulted from other inhibitors competitively binding with AChE and partly or completely displacing DDAO. Experimental results show that changes in fluorescence intensity are related to the concentration of inhibitors present in the solution. In addition, the nanobiosensor has high sensitivity, with detection limits as low as 0.4 μM for paraoxon and 10 nM for tacrine, and also exhibits different reduction efficiencies for the two types of inhibitor. Thus, instead of an inhibition test, a new type of affinity binding-guided fluorescent nanobiosensor was fabricated to detect AChE inhibitors, determine AChE inhibitor binding mode, and screen more potent AChE inhibitors. The proposed strategy may be applied to other proteins or protein domains via changes in the affinity ligand.
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challenge to position the particle in front of the molecule with nanometer accuracy. To date, a number of experimental methods have been developed for adjusting the distance between fluorophores and metal NPs in metal-enhanced fluorescence studies.6,12,13 All these methods mainly involved the design of metal-spacer-fluorophore hybrid structures, in which the distance between the fluorophores and metal NPs is adjusted by controlling the thickness of the spacer. The maximum fluorescence enhancements occur at an approximate distance readily obtained by one or two layers of proteins.12 Hence, the optimal distance for MEF can readily be obtained using protein monolayers. Acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses via rapid hydrolysis of the neurotransmitter acetylcholine (ACh). Many neurodegenerative diseases (Alzheimer’s disease, Parkinson disease, Huntington’s disease, etc.) are associated with the degeneration of the cholinergic system, resulting in a decrease in the amount of neurotransmitters, such as ACh. Inhibition of ACh hydrolysis
he surface plasmon resonance (SPR) of metallic nanostructures, especially silver and gold nanoparticles (AuNPs), can enhance the local electromagnetic field surrounding the NPs and ultimately lead to an increase in the fluorescence of nearby fluorophores.1,2 Using Au bowties, the observed enhancement of the fluorescence of a single molecule has been up to a factor of 1340.3 Recently, there has been a sudden increase in the use of metal-enhanced fluorescence (MEF) for its multifarious applications in medical diagnostics and biotechnological methodology, such as in immunoassays,4 protein translation,5 and DNA detection.6 Enhancement is attributed primarily to the increased electric field close to the metal NPs induced by incident light. Factors affecting these interactions include the electronic properties of the metal particle, the size of the NPs, coating material on NPs, the spatial separation of the fluorophore from the NPs surface, and the frequencies of the exciting and fluorescent radiation.7,8 The magnitude of fluorescence enhancement strongly depends on the distance between fluorophores and metal nanostructures. Maximum enhancement was found at distances of 7 nm− 10 nm9 and 5 nm−11 nm.10 Indeed, one of the complexities of MEF experiments is the achievement of distance control to obtain maximum enhancement.6,11 However, it is a great © 2012 American Chemical Society
Received: December 13, 2011 Accepted: February 17, 2012 Published: February 17, 2012 2830
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by blocking its metabolic enzyme AChE increases the ACh concentration and provides a symptomatic treatment option.14,15 AChE inhibitors that penetrate the blood-brain barrier have proved useful in the symptomatic treatment of Alzheimer’s disease. These are donepezil, rivastigmine, galanthamine, and tacrine.16,17 However, the complete inactivation of AChE, which can occur in organophosphate and carbamate insecticides 18 and organophosphate chemical warfare agents,18,19 leads to the toxic accumulation of ACh. In addition, failure of cholinergic synaptic transmission, with consequent deterioration of neuromuscular junctions, flaccid muscle paralysis, and seizures in the central nervous system, may also occur. Thus, interest in developing more accurate and sensitive methods for detection and determination of these AChE inhibitors has grown. Biosensors based on the principle of AChE inhibition have by now been applied to a wide range of irreversible inhibitors. These irreversible inhibitors include organophosphate and carbamate, in combination with potentiometric,20 amperometric,21,22 optical,23 and fluorimetric sensors.24 In general, the development of these biosensing systems relies on the quantitative measurement of enzyme activity before and after exposure to a target analyte. However, these inhibition-based sensors suffer from several limitations. One of the common problems of AChE inhibition-based biosensors is the percentage of inhibited enzyme activity. This inhibition which can also result from any environmental or handling factors that cause loss of enzyme activity will result in false positive signals. Another problem is the irreversibility of inhibition during analysis and the often impossible or impractical regeneration of the sensor. Relatively successful studies on the regeneration of inhibited AChE were published.25 Used sensors were usually discarded due to the decrease in their activities.26 Yet another problem is that the percentage of inhibited enzymes that results after exposure to the inhibitor is quantitatively related to the inhibitor (i.e., analyte) concentration and the incubation time.27 Consequently, the response signal is inversely related to the inhibitor concentration and the concentration of antianalyte association, resulting in difficulty in precise detection of low inhibitor concentrations. Several attempts were made to improve the performance of biosensors for AChE inhibitors, such as using recombinant AChE and artificial neural networks28 and employing piezoelectric affinity as sensing model.29 Lastly, residual activity is difficult to measure in reversible inhibitors because it is not easy to avoid inhibitor leaking, in which inhibitors are noncovalently bound to an enzyme. We describe an affinity binding-guided ligand-binding strategy to generate a fluorescent nanobiosensor geared toward detection and determination of the reversible and irreversible AChE inhibitors. The new concept is based on the distance modulation between the fluorophore and the metallic NP depending on the size of the AChE molecules. The fluorescence of DDAO (Figure S1) was sharply enhanced when DDAO bound to AChE conjugated with the AuNP as a result of MEF. The enhanced fluorescence of DDAO was then reduced due to combined functions of losing MEF of DDAO on AuNPs and increasing dynamic quenching of DDAO in the solution. This behavior resulted from distance variations between DDAO and AuNP because of DDAO disassociation caused by other AChE inhibitor binding.
Article
EXPERIMENTAL SECTION
Steady State DDAO Inhibition of AChE Catalyzed Substrate Hydrolysis. The activity of AChE was measured at 25 °C via the Ellman method.30 The assay mixture contained 1.2 mM 5,5′-dithiobis(2-nitro-benzoic acid) solution and 1.9 mM acetylthiocholine iodide in a 50 mM sodium phosphate buffer at pH 7.5. The mixture was examined spectrometrically at λ = 412 nm with the formation of the thiolate dianion of 5,5′dithiobis-(2-nitrobenzoic acid) for 1 min−2 min. During this interval, substrate concentrations were corrected for substrate depletion as a result of hydrolysis.31 DDAO was first dissolved in DMSO and then used for the experiment. The final concentration of DMSO in the test solution was 0.08% (v/v). Controls, without inhibitor but containing 0.08% DMSO, were routinely performed. The extent of inhibition with sample addition was expressed as the percentage necessary for 50% inhibition (IC50). The noncovalent inhibition of AChE is described in Supporting Information S2−1. Molecular Docking of DDAO with AChE. The SYBYL8.0 package (Tripos Inc. Web site. http://tripos.com) was used to prepare the protein and ligand data required for the docking experiments with Autodock 4.2.32 DDAO was originally drawn with Chem3D Ultra to obtain standard 3D structures. The ligand atom types and bond orders were corrected manually. Hydrogen atoms were added, and Gasteiger−Hückel charges were assigned.33 The geometries of the ligands were optimized for 200 steps with the Simplex algorithm using the Tripos force field34 and were saved in the SYBYL Mol2 format. The protein structure was obtained from the Protein Data Bank (PDB) (http://www.rcsb.org/) in .pdb format. Water molecules were removed from the surface of the protein, and all hydrogens were restored for ADT calculations. The Lamarckian Genetic Algorithm35 was used as the search engine. The active site was defined using AutoGrid. The grid size was set to 60 Å × 60 Å × 60 Å points with grid spacing of 0.375 Å centered on the ligand center of mass. The best conformation chosen was the lowest docked energy for the analysis of protein−ligand interactions, including hydrogen bonds and bond lengths. Fabrication of AuNP-AChE Bioconjugates. The AuNPAChE bioconjugates were fabricated according to the literature method36 with a slight modification. The citrate-stabilized AuNPs were freshly synthesized through the conventional method of reducing aurate chloride by sodium citrate in boiling solution.37 The subsequent MUA-modified AuNPs were prepared via ligand exchange between mercapto-carboxylic acid and citrate groups under the protection of nonionic surfactant Tween-20 (S2−2). The combination of AChE and MUA-modified AuNPs was based on the EDC/NHS coupling reaction with a slight modification.36 In detail, the above MUAmodified AuNPs were combined to react with a mixture of freshly prepared 50 mM NHS and 25 mM EDC solution for 10 min. Then, the NHS-terminated AuNPs were separated through centrifugation and resuspended in a phosphate buffer (10 mM, pH 6.8, with 0.2 mg/mL Tween-20) under ultrasonication for the subsequent wash. After discarding the supernatant, the remaining NHS-terminated AuNPs were incubated using 1.1 mg/mL AChE in a phosphate buffer (10 mM, pH 6.8) for more than 12 h under N2 atmosphere. The resultant mixture was centrifuged to remove free AChE and washed by phosphate buffer with Tween-20. The target AuNPAChE bioconjugates were finally dispersed under ultrasonication in a phosphate buffer (10 mM, pH 7.4) and stored 2831
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at 4 °C. The amount of AChE covalently immobilized with the AuNPs was measured at 25 °C via activity assay using the Ellman method as described above.30 Biosensing AChE Inhibitor via Fluorescence Measurement. Prior to experimental analysis, a stock solution of DDAO phosphate fluorophore was prepared at a concentration of 3 μM in deionized H2O. The emission spectra of DDAO were obtained within the spectral range of 575 to 750 nm using the maximal excitation wavelength at 447 nm. The maximal fluorescence intensity at 653 nm was determined. Typically, 7.5 μL of 1 nM of AuNP-AChE bioconjugate solution was added to the prepared 1 μM DDAO PBS buffer solution (1.00 mL, 10 mM, pH 7.4) for an equilibration period (30 min.) to react completely. Under this condition, the nanobiosensor AuNP− AChE−DDAO solution was formed and could be used for the subsequent AChE inhibitor sensing. The maximal fluorescence intensity of the AuNP−AChE−DDAO complex (F2) was determined again. Afterward, paraoxon or tacrine was added in different concentrations, and the maximal fluorescence intensity (F3) was measured at 15 min after reaction at room temperature. The maximal fluorescence intensity of the same amount of AChE-DDAO (F1) was used as a background signal level. Relative fluorescence intensity change ΔF was calculated as relative ΔF = (F3−F1)/(F2−F1). This represents the ratio of the enhancement of fluorescence in the presence of paraoxon or tacrine (Figure S1) to the enhancement of fluorescence in the absence of paraoxon or tacrine. Control experiments were performed to check any fluorescence intensity changes in the absence of AuNP-AChE.
Figure 1. Schematic illustration of the strategy for the “on-off” type of fluorescent nanobiosensors for AChE inhibitors. (a) Fabrication of AChE-AuNP conjugate via EDC/NHS coupling chemistry. (b) Metal enhanced fluorescence of DDAO bound with the active sites of AChE via distance regulation between the fluorophore and AuNP. (c) The fluorescence reduction via distance modulation due to AChE inhibitor binding.
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AuNP, its fluorescence intensity is reduced. AChE plays a dual role hereit not only acts as spacer molecules to modulate the distance between fluorophore and metal surface but also as receptor for the binding affinity of the fluorophore and AChE inhibitor. Therefore, instead of inhibition test, the binding affinity properties of AChE were directly applied to its detection and determination. Kinetic Evaluation of DDAO Binding with AChE. The kinetic inhibition mechanism of DDAO on AChE was first studied to obtain information on the binding mode of DDAO. Steady-state inhibition data were graphically analyzed by Lineweaver−Burk plots, and the results are illustrated in Figure 2. Double-reciprocal plots yielded a group of lines intersecting at the second quadrant; the y-intercept corresponds to the reciprocal of maximal velocity (Vm ), while the slope corresponds to the reciprocal of Vm/Km. With the increase in concentration of DDAO, Michaelis constants (Km) increased and Vm decreased. These results show the occurrence of a reversible competitive-uncompetitive mixed type-I AChE inhibition (S2−1).30 This type of inhibition can be very attractive in terms of the ability of the molecule to bind to the peripheral anionic site of the AChE. The molecule produced a concentration-dependent AChE inhibitory effect with an IC50 value of 2.50 × 10−7 M, which is very close to the calculated theoretical IC50 value of 2.52 × 10−7 M (S5, Figure S3). The calculated KI and KIS are 1.70 ± 0.25 × 10−7 M and 6.74 ± 0.21 × 10−7 M, respectively (Figure 2). The value of KIS is about four times as high as KI, indicating that the affinity of the inhibitor for the free enzyme is stronger than that of the inhibitor for the enzyme−substrate complex.42 Molecular Docking of DDAO-Binding with AChE. To propose a plausible binding orientation and bioactive conformation of the AChE-DDAO complex that may explain the observed experimental inhibitory activity and illustrate the
RESULTS AND DISCUSSION General Design of Affinity-Guided Labeling and Sensing. Our idea was to use the size and binding properties of AChE and construct a fluorescent biosensor to directly monitor the binding affinity of its inhibitor. The concept was based on maximum fluorescence enhancements which occur from an approximate distance 5 nm−11 nm from the metal surface. By taking advantage of this strategy, as illustrated in Figure 1, AChE molecules were covalently bound to the AuNP via MUA and EDC/NHS chemistry. The first crystal structure, that of Torpedo californica AChE (TcAChE), was determined in 1991.38 TcAChE was seen to be an ellipsoid with dimensions ∼45 Å × 60 Å × 65 Å. Ligand-binding studies and X-ray crystallography have revealed that the active site of AChE contains three key motifs:38−41 an active-site (A-site), a peripheral anionic site (P-site), and a long narrow hydrophobic gorge connecting the A- and P-sites. The A-site contains a catalytically reactive serine located 20 Å from the protein surface and is the principal target for noncovalent inhibitors such as tacrine and acridine and covalent modifiers such as organophosphates and carbamates.42,43 The P-site is located at the protein perimeter and contributes to catalytic efficiency by transiently binding substrates on their way to the A-site.39 The gorge is lined with hydrophobic residue to facilitate passage of substrate and other molecules (S4, Figure S2).41 The fluorophore DDAO was found to be a reversible AChE inhibitor and can serve as ligand bound to the P-site of AChE. Once the fluorophore is introduced to the solution, it can bind with and be labeled to AChE, which conjugated with the metallic NP. Thus, its fluorescence emission is enhanced by the metallic NP. When the AChE inhibitor binds to the active site of AChE, the fluorophore bound to the enzyme will be displaced and move away from the 2832
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Figure 2. Lineweaver−Burk plot for the determination of the inhibitory mechanism of DDAO on AChE. (a) The concentrations of DDAO for curves 1−5 were 0, 3, 6, 9, and 12 × 10−8 M, respectively. (b) and (c) represent the plot of slope and intercept versus the concentration of DDAO for determining the inhibition constants KI and KIS, respectively.
binding mode, the ability of DDAO to interact with AChE was further assessed via in silico studies. Figure 3a depicts the molecule docked at the active gorge site; Autodock 4.2 software was used for the conformer with the lowest calculated binding energy of −28.36 kJ/mol. A visual inspection of the highestscoring docking conformation for DDAO revealed a recurrent configuration. The 7-hydroxy moiety of DDAO was located at the peripheral anionic site near the mouth of the active narrow gorge, where it formed a hydrogen bond with the carbonyl group of Asp72 (Figure 3). Moreover, considering that the acridine ring of DDAO was found occupying the gorge, this molecular region was likely to be completely buried inside the enzyme. Figure 3b depicts the binding mode of DDAO. The important residues for DDAO binding and/or catalysis are present at the binding site (Asp72, Trp84, Phe290, Phe330, Asn83, Gly117, and Gly118).41 A strong hydrogen bond is established between the oxygen atom of the 7-hydroxy moiety of DDAO and the Asp72 (O−O distance of 2.8 Å) residue. This hydrogen bond, as well as the π−π stacking interaction between the acridine ring of DDAO and the indole of Trp84, is proposed to occur during the primary binding to the active site. According to these computational studies, DDAO can bind to the peripheral anionic site of AChE but is able to penetrate into the enzyme active site gorge as a secondary binding site. This finding is similar to what has been reported for tacrine, which was investigated by X-ray crystallography and determined at 2.8 Å.42 The docking result is consistent with the result of the inhibition kinetics test, showing that the competitive effect is stronger than the uncompetitive effect. Fabrication of AuNP-AChE Bioconjugates. The intense Au−S covalent bond formed because AuNPs have an extremely high affinity toward thiols and thiol-modified molecules. The AuNP-AChE bioconjugates were constructed using the method for AuNP-glucose oxidase bioconjugates reported by Li et al.36 with a slight modification. As a typical prescription, the concentration of colloidal Au was approximately 0.8 nM. The measured activity of AuNP-AChE conjugate was 0.2 U/mL, the specific activity of free AChE for immobilization was 420 U/ mg, and the molecular weight of AChE monomer is 70 kDa.
Figure 3. Molecular docking of DDAO with TcAChE. (a). Superposition of the docked conformations of DDAO in the TcAChE binding site. (b) Binding mode of DDAO in the TcAChE active site. The important residues for DDAO binding in the TcAChE active site, according to visual inspection, considering the amino acid residues in a radius of 6 Å around the ligand. Broken lines indicate hydrogen bonds.
Therefore, the calculated number of AChE covalently bound to the NPs was 8 AChE monomer molecules per AuNP. The results from TEM images and UV−vis spectra show that such bioconjugates still maintain a relatively good dispersity (Figure 4, S7). The enzyme thermostability and pH-dependence of such assembled bioconjugates were also tested (S2−3). The AuNP-AChE bioconjugates can hold the enzyme activity at different pH values and are as stable as a free enzyme in solution against high temperature (Figure S4). These results show that the AChE molecules conjugated well with the AuNP and maintained their natural forms. MEF and Biosensing AChE Inhibitors. After the AChE molecules were immobilized onto the surface of the AuNPs through covalent bonds, the excess free AChE was removed via centrifugation and washing of the AuNPs-AChE conjugates in small aliquots. Then, the AuNP-AChE conjugates were resuspended in buffer. The concentration of AuNP-AChE conjugates in the solution was determined by AChE activity assay. As described above, we determined that eight AChE molecules were conjugated to each AuNP. When AuNP was set as reference, the final concentration of AuNP(AChE)8 conjugates in buffer was adjusted to 1 nM for further use. 2833
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Figure 5. Emission spectra of AuNP-AChE-DDAO bioconjugates in the presence of various concentrations of (a) paraoxon and (b) tacrine and the dynamic ranges (insets). The final concentration of AuNPAChE conjugate is 1 nM. λex = 447 nm. (a): 1, 1 μM DDAO; 2, 1 μM DDAO with AChE; 13, AChE-AuNP bioconjugate containing 1 μM DDAO; 12−3, AChE-AuNP bioconjugate containing 1 μM DDAO with final concentrations of paraoxon from 12 to 3 are 0.5, 1.0, 2.5, 4.0, 5.0, 6.5, 8.0, 10.0, 15.0, and 20.0 μM, respectively. (b): 1, 1 μM DDAO; 4, 1 μM DDAO with AChE; 13, AChE-AuNP bioconjugate containing 1 μM DDAO; 12−5, AChE-AuNP bioconjugate containing 1 μM DDAO with final concentrations of tacrine from 12 to 5 are 15, 20, 35, 50, 65, 80, 100, and 120 nM; 3 and 2 are 150 and 200 nM, respectively. The inset dynamic ranges are shown as the relative fluorescence intensity change (relative ΔF) vs final paraoxon (a) and tacrine (b) concentration.
Figure 4. TEM images and UV−vis spectra of (1) citrate-stabilized AuNPs, (2) MUA modified AuNPs, (3) NHS-terminated AuNPs, and (4) the AChE-AuNP bioconjugates.
The AuNP-AChE-DDAO conjugation was performed at room temperature. Given that the DDAO is a reversible AChE inhibitor, excess DDAO was added to saturate the AuNP-AChE conjugate. Rather than DDAO, AChE-DDAO was used as the control sample in aqueous solution to ensure that the fluorophore was in essentially the same chemical environment in all the measurements. The reference sample, as well as the NP conjugates, was excited at 447 nm. The fluorescence spectra were obtained in a solution under identical excitation and detection conditions, allowing direct comparison of the various AuNP-AChE-DDAO complexes. A maximum fluorescence enhancement of ∼6-fold was measured at 653 nm (Figure 5). In contrast to DDAO phosphate as a substrate of organophosphate hydrolase,44 DDAO is not a substrate of AChE but a ligand. DDAO can form stable complex with AChE and not be hydrolyzed by AChE during assay. Thus, a more stable signal response and less background effect were obtained. Additionally, because the fluorescent molecule is the ligand of the protein itself, the conjugate fabrication does not need to covalently introduce fluorescent molecules via chemical modification. It is well-known that chemical modification has several drawbacks: 45 the appropriate site and type of fluorophore to be introduced to achieve the optimal fluorescence response is difficult to predict; protein denaturation may occur because of the multiple chemical reactions to be
performed on the protein surfaces; and a careful purification step is also required after the labeling reaction. Control experiments were performed to evaluate the influence of either paraoxon or tacrine on the fluorescence intensity of DDAO and DDAO bound with AChE, respectively. Only minor differences in fluorescence intensity between DDAO and DDAO plus paraoxon were observed; it could be calibrated under the same test condition. The fluorescence of DDAO can be slightly enhanced by the same amount of free AChE as the immobilized AChE with AuNPs. This slight enhancement was subtracted as background signal as described in the method section. To test the feasibility of biosensing for irreversible AChE inhibitors, we initially tested the AuNP-AChE-DDAO conjugate for biosensing paraoxon. The primary mechanism of action for paraoxon, commonly used as organophosphorus insecticides, is to inhibit AChE due to the phosphorylation of 2834
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On the other hand, paraoxon was covalently bound to ser200, partly overlaid with the binding site DDAO. Therefore, attributing to the maximum reduction efficiency, the method can determine the binding mode of different inhibitors.
the serine hydroxyl group located in the active site of the molecule.46 Replacements of residues Phe-295 and Phe-297, constituting the human AChE acyl pocket, increase up to 80fold the reactivity of the enzymes, indicating the role of this subsite in accommodating the phosphate alkoxy substituent.43 The AuNP-AChE conjugates were prepared and then incubated with DDAO followed by the enhancement (“switch-on”) of DDAO fluorescence. The resulting AuNP-AChE-DDAO conjugates can be used directly for fluorescence assay without the need of any additional step. Upon the introduction of paraoxon into the sensing system, paraoxon occupies competitively the acyl pocket on AChE, which was originally combined with DDAO. DDAO was then displaced from the acyl pocket on AChE followed by the reduction (“switch-off”) of fluorescence. We observed a regular decrease in the fluorescence intensity of the AuNP-AChE-DDAO in response to different concentrations of paraoxon (Figure 5a). A plot of relative ΔF versus paraoxon produced a straight line in the concentration range of 0.5 μM−10 μM (Figure 5a, inset). Statistical analysis reveals a detection limit of paraoxon concentration of as low as 0.4 μM. The precision was expressed as the relative standard deviation (%RSD). The %RSD obtained from a series of 11 standard samples each containing 0.5 μM of paraoxon was 1.7. Therefore, the sensitive detection of irreversible AChE inhibitors via the newly assembled nanobiosensor is feasible. We then tested the feasibility of the AuNP-AChE-DDAO conjugate for biosensing, tacrine, a well-known reversible AChE inhibitor currently approved for the treatment of Alzheimer’s disease.47 The binding sites of TcAChE for tacrine were investigated via X-ray crystallography and determined at 2.8 Å resolution (PDB code 1ACJ).42 Tacrine cocomplexed with TcAChE at the active site gorge, where its amino nitrogen formed a hydrogen bond to a water molecule. The acridine was stacked against the indole of Trp84, and its ring nitrogen formed a hydrogen-bond with the main-chain carbonyl oxygen of His-440.42 The same sets of sensing experiments were performed for the detection of tacrine. A regular decrease in the fluorescence intensity of the AuNP-AChE-DDAO in response to different concentrations of tacrine was also observed (Figure 5b). The relative change in fluorescence intensity was correlated with the concentration of tacrine present in the solution, with a dynamic range from 15 nM to 120 nM (Figure 5b, inset). The detection limit and determined %RSD of the tacrine determination were 10 nM and 2.1, respectively. Consequently, the sensing system was more sensitive to tacrine compared with paraoxon. Although tacrine is a reversible inhibitor, it can strongly bind with both AChE and AChEsubstrate complex. The determined KI and KIS of the tacrineTcAChE and tacrine-TcAChE-substrate complexes are 6.4 and 7.4 nM at 0.1 M NaCl at 1:1 binding stoichiometry, respectively.48,49 In this case, the percentage ratio of the maximal fluorescence intensities can reach approximately 100% in comparison with DDAO solution as the tacrine concentration increased. The fluorescence intensity of this sensing system at the highest tacrine concentration test was less than that of AChE-DDAO solution. On the contrary, even at the highest paraoxon concentration tested, the percentage ratio of the maximal fluorescence intensities can reach approximately 145% and 170% of that of AChE-DDAO and DDAO solution, respectively (Figure 5). The reason is that the binding site of tacrine with AChE is more consistent with DDAO. As a result, tacrine displaced DDAO from the conjugate more efficiently.
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CONCLUSION The presented AChE-based fluorescent biosensor, which consists of a natural protein scaffold and artificial fluorescent molecule, is sensitive for the direct detection of AChE inhibitors. The biosensor can determine the binding characteristics of these inhibitors as well. The protein scaffolds functioned not only as a distance modulator between the fluorophore and metallic NP but also as a receptor for competitive ligand-binding between the fluorophore and inhibitors. Thus, the distance between the metallic NP and the fluorophore can be controlled by the AChE molecule to achieve efficient fluorescence enhancement. The detection of the inhibitor is not based on the inhibition of AChE activity but on the displacement of the fluorescent ligand by the AChE inhibitor. Therefore, the enzyme activity need not be measured to avoid false positive results, which originated from reduction of the activity by other factors. Additionally, because the present biosensing system is a competitive assay, it can set an appropriate threshold to eliminate weak binding ligands, which is generally advantageous for the screening of a more potent binder.50 The fluorescence-enhanced factor can be further improved by reducing the background signal through the adjustment of the amount of fluorescent ligand in the system or by optimizing the NP topology, dimensions, and composition.51 The proposed strategy can be applied to monitor the interaction of other proteins or protein domains with their ligands using their affinity-fluorescent ligands for high throughput drug screening before enzymatic inhibition assay is carried out to verify the inhibition of the newly found ligands. Investigations into these topics are now underway.
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ASSOCIATED CONTENT
* Supporting Information S
Text, Figures S1−S4, and Scheme S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86-29-81530726. Fax: +86-29-81530727. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are indebted to Prof. Dr. C. X. Zhang, X. W. Zheng, Q. Gao, H. L. Qi, and Y. Jin for their kind help and valuable suggestions. This work was supported by Program for Changjiang Scholars and Innovative Research Team in University (IRT 1070), the National Natural Science Foundation of China (30600494), and the Fundamental Research Fund for the Central Universities (GK200902010).
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REFERENCES
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Analytical Chemistry
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dx.doi.org/10.1021/ac300436m | Anal. Chem. 2012, 84, 2830−2836
