Aptamer-based electrochemical biosensor for Botulinum neurotoxin
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We have developed an aptamer-based electrochemical sensor for detection of Botulinum neurotoxin, where steric hindrance is applied to achieve specific signal amplification via conformational change of the aptamer. The incubation time and potassium concentration of the reaction buffer were found to be key parameters affecting the sensitivity of detection of the recognition of Botulinum neurotoxin by the aptamer. Under optimized experimental conditions, a high signal-to-noise ratio was obtained within 24 h with a limit of detection (LOD) of 40 pg/ml by two standard deviation cutoffs above the noise level.
KeywordsAptamer Botulinum neurotoxin Biosafety Electrochemical biosensors Steric hindrance Conducting polymer Bioanalytical methods Bioassays Biosensors
Botulinum neurotoxin (BoNT) is among the most toxic substances known [1, 2]. Modeling studies shows 4 g per gallon of BoNT in milk could cause rapid distribution more than 400,000 causalities within days . Therefore, rapid and sensitive detection of BoNT are highly desirable for addressing biosafety concerns. Among the seven defined serotypes of BoNT (A to G), type A is the most frequent cause of cases of human botulism . Conventional methods for detection of BoNT are primarily immunoassay-based and have a sensitivity of 2–60 pg/ml [5, 6]. However, immunoassay of BoNT requires time-consuming and labor-intensive process of screening highly specific human monoclonal antibodies [5, 7]. In contrast, aptamers are fragments of oligonucleotides which can bind specifically to target proteins in the presence of appropriate conformational change [8, 9, 10] and have the advantages of simpler screening methods, higher stability than antibodies and reusability. Recently, an efficient single micro-bead SELEX approach has been developed to generate high-affinity single-stranded DNA (ssDNA) aptamers against aldehyde-inactivated Botulinum neurotoxin type A (BoNT/A) . The individual dissociation constants (Kds) range from 3 nM to 50 nM for different aptamer designs.
Here, we propose a strategy that combines an electrochemical method [12, 13, 14, 15] with enzymatic amplification and an aptamer probe that undergoes target-induced conformational change for the detection of BoNT/A toxoid (BoNT/A). The electrochemical sensor has a streptavidin-dendrimer-interfaced polypyrrole substrate to maintain the activity of the aptamer and BoNT/A. The aptamer is dual-labeled with a reporting tag (fluorescein) and an anchoring tag (biotin). In the signal amplification process, an anti-fluorescein antibody conjugated to horseradish peroxidase (HRP) is introduced to bind to the fluorescein label on the aptamer. After antibody binding 3, 3′, 5, 5′ tetramethylbenzidine (TMB/H2O2) is added for the signal readout under −200 mV potential. TMB acts as a mediator and is reduced at −200 mV; the reduced TMB reduces the oxidized form of HRP. HRP then reduces H2O2 to 2H2O and the HRP is oxidized. This signal amplification process only occurs under conditions that permit HRP binding to the aptamer on the surface. In the absence of BoNT/A binding, the aptamer remains in the closed state and steric hindrance [16, 17] from the sensor surface inhibits signal amplification by preventing anti-fluorescein-HRP from accessing the reporting tag fluorescein. After binding to BoNT/A, the aptamer conformation opens up and permits access of the HRP reporter, generating an electrochemical current signal. Therefore, only the specific target BoNT/A can generate an amplified current.
Materials and methods
Aptamer and BoNT/A toxoid
HPLC-purified aptamer oligonucleotides, biotin-labeled on the 5′ end and fluorescein-labeled on the 3′ end, were custom-synthesized (Operon Inc., Alabama, USA). Biotin bound to surface streptavidin served as an anchor to the electrode and the fluorescein label allowed for binding of anti-fluorescein-HRP to amplify the electrochemical current signal.
The 76-bp aptamer sequence for BoNT/A detection  was 5′-ATACCAGCTTATTCAATT GAC ATG ACT GGG ATT TTT GGC GAA ATC GAA GGA AGC GGA GAGATAGTAAGTGCAATCT-3′.
G-quadruplex 1: GGGATTTTTGGCGAAATCGAAGGAAGCGG
G-quadruplex 2: GGATTTTTGGCGAAATCGAAGGAAGCGG
BoNT/A toxoid (toxin inactivated by formaldehyde treatment) with a molecular weight of 150 kDa was purchased from Metabiologics, Inc (Madison, WI).
Surface fabrication of streptavidin-polypyrrole electrodes
The electrode surface consisted of a conducting layer of streptavidin-coated polypyrrole (PPy) polymerized on gold thin film. Streptavidin was introduced into the PPy matrix by copolymerization of a streptavidin-modified DNA dendrimer (Genisphere, USA, diameter 70–90 nm) and pyrrole (Sigma, USA). The streptavidin-modified DNA dendrimer was composed of short oligonucleotides and could be easily incorporated into the PPy matrix due to the negative charge of the dendrimer. Each dendrimer was labeled with two to four streptavidin molecules.
For electropolymerization, the streptavidin-labeled dendrimer was diluted with pyrrole in 1× PBS (pH 7.5, Invitrogen, USA) at a ratio of 1:200 (v/v) for a final concentration of 10 mM pyrrole. The electrodes were covered with the mixture of dendrimer and pyrrole prior electropolymerization. To avoid exposure of the dendrimer to a strong electrical field and to form a smooth polymer surface, square-wave electrical field was applied for electropolymerization . Each square-wave consisted of 9 s at a potential of +350 mV and 1 s at +950 mV. A total of 20 cycles of square-waves was applied and the entire process lasted for 200 s. After polymerization, the electrode was rinsed with ultra pure water (18.3 MΩcm) and dried under a stream of pure N2. The polymer film was 51.5 ± 3.0 nm thick, as measured by a profilemeter (Dektak 6 Surface Profile Measuring System, Veeco). The surface concentration of streptavidin-modified dendrimer was 0.30 ± 0.05 pmol/cm2, as measured with a scanning electron microscope (Hitachi S4700 SEM, Japan).
The electrochemical sensor consisted of a 16-unit gold array. Each unit was comprised of three electrodes, including the working electrode (WE), counter electrode (CE), and reference electrode (RE) [17, 22]. The reference electrode was determined to be +218 mV vs. SCE by measuring cyclic voltammetric curves of 0.1 mM [Fe(CN)6]3−/4−. All electric potentials described in this report are in reference to the gold reference electrode (+218 mV vs. SCE) .
In-solution or surface-associated folding of the BoNT/A aptamer
Detection of BoNT is based on the signal generated by the conformational change of the aptamer. Correct folding of the aptamer allows high-affinity binding between the aptamer and BoNT/A toxoid; misfolding and non-folding result in low signal-to-noise ratios (SNRs) . In addition, the specific signal amplification is strongly dependent on the conformation of the aptamer before and after binding to BoNT/A. Strong signal is generated by completely unfolded aptamer binding to target BoNT/A and low noise occurs in the presence of fully folded aptamer in the absence of target. Therefore, understanding how to obtain correct folding of aptamer is key to the detection process.
Figure 3 shows that the signal generated by recognition in solution is approximately 900 nA higher than that generated by recognition on the surface. The SNR is 6.8 for in-solution recognition and 1.3 for the surface recognition. Background levels are low in both processes (observed differences in background may be the result of the different buffers used in the immobilization and recognition processes). With the low surface concentration of streptavidin (0.30 ± 0.05 pmol/cm2), crowding effect is not the critical problem for aptamer surface binding. Since the concentrations of aptamer and BoNT/A toxoid are identical in the two processes, the only difference is the medium in which recognition takes place. As discussed earlier, the SNR depends on the correct folding of the aptamer. Therefore, the aptamer in solution has higher binding and self-folding efficiency than the aptamer immobilized on the surface. One possible explanation for this phenomenon is that the aptamer in solution can fold freely and has a greater chance of interacting with target BoNT/A toxoid in three dimensions, resulting in a higher possibility of correctly folding and docking with target. However, folding of the aptamer on the surface of the electrode may be constrained by chemical or physical interactions between the aptamer and the surface; docking with the target toxoid may be similarly restricted. Based on the assumption that attachment of the aptamer to the surface hinders the recognition of aptamer and target toxoid in this manner, the remaining experiments were carried out using recognition in solution.
Folding time and potassium concentration in aptamer-based BoNT/A detection
Four different incubation times were investigated for aptamer folding in in-solution recognition.
Amperometric detection of BoNT/A toxoid following different incubation times
Incubation time (hours)
−376 ± 64
−256 ± 26
−1142 ± 197
−168 ± 5
−1204 ± 100
−100 ± 2
−1986 ± 42
−52 ± 6
Here, we report an aptamer-based BoNT/A detection system that displays specific signal amplification and a high SNR and is based on conformational change in the aptamer induced by steric hindrance. This steric hindrance is caused by either the opened or closed conformation of the aptamer [17, 33]. When the aptamer binds to target BoNT/A toxoid it is in its open state and signal increases. If the target is not bound, the aptamer remains in its closed conformation, resulting in low signal. Unlike a traditional hairpin probe, which is designed with an inner stem region so it can be triggered to open or close, this specific BoNT/A aptamer does not contain such a “switch” in the sequence. However, this aptamer has a relatively long sequence of 76 bp and two interior G-quadruplexes. Therefore, the aptamer will be in a tightly coiled, or closed, state in the absence of BoNT; after binding to BoNT conformational change occurs. Most aptamer–protein binding events consist of multi-point docking or surface–surface interactions, rather than simple point–point docking. This complex docking process results in the open conformation of the BoNT/A aptamer.
In our optimization of incubation time, we observed that short incubation times resulted in very low SNRs, while longer incubation times resulted in high SNRs. One possible explanation for this observation is based on the reaction energy. A high energy barrier would exist and several misfoldings would occur during the reaction process, resulting in different quasi-stable intermediates. Although the appropriate configuration is the most stable one and has the lowest Gibbs free energy, trapping in the quasi-stable intermediates can occur. In our investigation, short durations of folding (less than 3 h of incubation) generated an SNR of only 1.5, while longer-duration (greater than 5 h) incubations generated SNRs of 12 or greater (SNR of 38 after 24 h of incubation). This data provides evidence supporting the existence of quasi-stable, misfolded conformational intermediates prior to achievement of the final stable conformation.
To drive the reaction toward the thermodynamically stable product, an important factor for correct folding is the ion composition of the reaction buffer. Our data show that the affinity of the aptamer for BoNT/A is dependent on the potassium ion composition of the buffer, with a concentration of 15 mM KCl generating the highest SNR. With the potassium concentration optimized, the aptamer binds to BoNT/A with high affinity and the LOD of the sensor is 40 pg/mL (400 fM). Other metal ions in addition to K+ should also affect the folding of aptamer, including Ca2+, Mg2+, Na+. Meanwhile, the folding process is also influenced by more than a single type of ion and future work will investigate optimized concentrations of combinations of multiple types of ions, such as sodium, potassium, magnesium, and calcium.
Steric hindrance resulting from the structure of the aptamer introduces specific and high signal after the aptamer binds to BoNT/A toxoid. The binding affinity of the aptamer for BoNT/A toxoid depends on correct folding of the aptamer. Optimized incubation time and potassium ion concentration results in a high signal-to-noise ratio. The LOD is 40 pg/ml under optimized conditions within 24 h, which is much shorter than the current record, 4 days.
This work was supported by funds from the NASA National Space Biomedical Research Institute (TD00406), the Pacific-Southwest Center for Biodefense and Emerging Infectious Diseases Research UC Irvine/NIH NIAID Award (1 U54 AI 065359) 2005–1609, National Institutes of Health/National Institute of Dental and Craniofacial Research (UO1DE 017790, UO1DE015018, and RO1DE017593) and the National Institutes of Health Nanomedicine Roadmap (Center for Cell Control, PN2EY018228).
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