Letter pubs.acs.org/ac
Aptamer-Based Viability Impedimetric Sensor for Bacteria Mahmoud Labib,† Anna S. Zamay,§ Olga S. Kolovskaya,‡ Irina T. Reshetneva,§ Galina S. Zamay,‡ Richard J. Kibbee,⊥ Syed A. Sattar,⊥ Tatiana N. Zamay,‡ and Maxim V. Berezovski*,† †
Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5, Canada Institute of Molecular Medicine and Pathological Biochemistry, Krasnoyarsk State Medical University, 1 P. Zheleznyaka str., Krasnoyarsk 660022, Russia § Department of Microbiology, Krasnoyarsk State Medical University, 1 P. Zheleznyaka str., Krasnoyarsk 660022, Russia ⊥ Centre for Research on Environmental Microbiology, Faculty of Medicine, University of Ottawa, 451Smyth Road, Ottawa, Ontario K1H 8M5, Canada ‡
S Supporting Information *
ABSTRACT: The development of an aptamer-based viability impedimetric sensor for bacteria (AptaVISens-B) is presented. Highly specific DNA aptamers to live Salmonella typhimurium were selected via the cell-systematic evolution of ligands by exponential enrichment (SELEX) technique. Twelve rounds of selection were performed; each comprises a positive selection step against viable S. typhimurium and a negative selection step against heat killed S. typhimurium and a mixture of related pathogens, including Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Citrobacter f reundii to ensure the species specificity of the selected aptamers. The DNA sequence showing the highest binding affinity to the bacteria was further integrated into an impedimetric sensor via self-assembly onto a gold nanoparticle-modified screen-printed carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect S. typhimurium down to 600 CFU mL−1 (equivalent to 18 live cells in 30 μL of assay volume) and distinguish it from other Salmonella species, including S. enteritidis and S. choleraesuis. This report is envisaged to open a new venue for the aptamer-based viability sensing of a variety of microorganisms, particularly viable but nonculturable (VBNC) bacteria, using a rapid, economic, and label-free electrochemical platform.
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immunosensor for enrichment and specific capture of viable Escherichia coli O157:H7 using a quartz crystal microbalancebased sensor was recently reported.8 However, the assay is sophisticated and takes up to 24 h for detection which limits time-to-results performance. Detection of viable E. coli JM101 was also achieved using a mass-change sensitive cantilever sensor and a probe, 2′,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), that accumulates only in live cells inducing a mass-change response to determine the cell viability in a short time.9 However, the assay is not label-free and lacks sensitivity (limit of detection, LOD ∼ 2000 viable cells) because of its dependence on a mass-sensitive sensor. Electrochemical impedance spectroscopy (EIS) is a technique generating more interest for biologists as it has a less destructive effect on the measured biological interactions because it is label-free and is performed at a very narrow range of small potential.10,11 This method was successfully employed to monitor viable bacteria by measuring the permittivity of the medium containing them.12,13 Unfortu-
almonellosis is a serious health concern and a major cause of many food poisoning cases worldwide. A number of foodborne illness outbreaks have occurred where the source of contamination has been traced to Salmonella typhimurium, which is recognized as the second common serotype (after S. enteritidis) found in humans.1 The conventional bacteriological method used to assess the presence of the pathogen and to determine whether it is alive or dead (ISO 6579 std.) is based on pre-enrichment in a nonselective media, followed by selective plating and subsequent biochemical and serological confirmation, which takes 2−3 days for presumptive results and up to 7−10 days for confirmation.2 This method is timeconsuming, labor-intensive, and is not valid for detection of viable but nonculturable (VBNC) strains of Salmonella.3 Detection of prokaryotic mRNA was previously reported as an indication for bacterial viability.4,5 However, this method requires pretreatment steps to condition the test samples followed by cell lysis to extract the target mRNA. Detection of live bacteria was achieved using potentiometric sensors6 and sensors based on functional polymers that can capture only living aerobic and facultative anaerobic microorganisms.7 However, the restriction of detection to aerobes represents the major drawback of these methods. A piezoelectric © 2012 American Chemical Society
Received: October 10, 2012 Accepted: October 17, 2012 Published: October 17, 2012 8966
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affinity to the bacteria were sequenced; the sequences are provided in Table S1, Supporting Information. An ssDNA sequence (STYP-3, GAG TTA ATC AAT ACA AGG CGG GAA CAT CCT TGG CGG TGC) showing the highest affinity to S. typhimurium was modified at the 5′ position with a 6hydroxyhexyl disulfide group (Integrated DNA Technologies).
Figure 1. Schematic diagram of the aptamer-mediated electrochemical detection of live Salmonella typhimurium bacteria.
nately, the measured signals suffer from interference of the electric properties of the background electrolyte. This effect is frequency dependent and cannot be reliably subtracted.14 Recently, we have developed an aptamer-based viability sensor for viruses (AptaVISens-V),15 taking advantage of the tunable specificity of aptamers which enabled the sensor to distinguish between viable and nonviable vaccinia viruses. In the present work, DNA aptamers specific to live S. typhimurium bacteria were selected on the basis of cell-systematic evolution of ligands by exponential enrichment (SELEX)16 from 80nt DNA library containing 40 nt random region, 5′-CTC CTC TGA CTG TAA CCA CG N40 GC ATA GGT AGT CCA GAA GCC-3′ (Integrated DNA Technologies, U.S.), as provided in detail in the Supporting Information. Aptamer selection consisted of 12 rounds; each comprising alternating negative- and positive-selection steps except the first round where only positive selection was adopted to enrich the ssDNA pool with binding aptamers. Positive selection was performed against live S. typhimurium to increase the affinity of the aptamers, whereas negative selection was carried out against heat-killed S. typhimurium and a mixture of related pathogens, including S. enteritidis, E. coli, S. aureus, P. aeruginosa, and C. f reundii to increase the selectivity of the aptamers. Briefly, the first round of selection (only positive) comprised 4 steps, including (1) incubation of the ssDNA library with viable S. typhimurium, (2) removal of unbound aptamers, (3) extraction of bound aptamers, and (4) amplification of extracted aptamers using symmetric and asymmetric PCR. The next eleven rounds of selection were carried out starting from the negative selection and comprised 6 steps, including (1) incubation of the ssDNA sequences obtained from the previous step with heat-killed S. typhimurium and a mixture of related pathogens and (2) collection of unbound aptamers which was followed by the previous four steps employed for positive selection. The affinity of the collected aptamer pools to S. typhimurium was analyzed by flow cytometry. This was performed by incubation of 100 nM of Alexa-488-labeled aptamer pools with 50 × 103 colony-forming units (CFU) of the bacteria followed by flow cytometric analysis. It was observed that the aptamer pool collected at the seventh round of selection had the highest binding affinity to the bacteria (KD = 25 nM). Subsequently, this pool was cloned, and the clones showing the highest
Figure 2. (A) Cyclic voltammograms of the viability impedimetric sensor after each immobilization or binding step. Cyclic voltammograms were recorded at a scan rate of 100 mV s,−1 where (a) bare SPGE; (b) after self-assembly of the thiolated S. typhimurium-specific aptamer; (c) after backfilling with 0.1 mM 2-mercaptoethanol. (B) Nyquist plot (−Zim vs Zre) of impedance spectra obtained using (a) 1 × 103, (b) 0.5 × 104, (c) 1 × 104, (d) 0.5 × 105, and (e) 1 × 105 CFU mL−1 of S. typhimurium in Dulbecco’s phosphate buffered saline (DPBS). The right inset represents a modified Randles circuit applied to fit to the measured data. The left inset represents a calibration plot of the resistance to charge transfer (RCT) vs log concentration of S. typhimurium. The impedance spectra are recorded from 100 kHz to 0.1 Hz, and the amplitude is 0.25 V vs pseudo Ag reference using 20 mM Tris-ClO4 buffer (pH 8.6), containing 2.5 mM K4Fe(CN)6 and 2.5 mM K3Fe(CN)6. 8967
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indicating the formation of a highly compact layer (curve b). Final treatment with 2-mercaptoethanol significantly reduced the redox currents because they could penetrate down to the electrode surface, thereby blocking the direct access of the conducting ions (curve c). Prior to titration experiments, aliquots containing different concentrations of S. typhimurium (1 × 103, 0.5 × 104, 1 × 104, 0.5 × 105, and 1 × 105 CFU mL−1) in 30 μL of Dulbecco’s phosphate buffered saline (DPBS, #D8662, Sigma-Aldrich, U.S.) were incubated with the aptasensor at 25 °C for 1 h. Electrochemical impedance spectroscopy (EIS) was performed at each concentration (Figure 2B). The complex impedance was presented as the sum of the real Z, Zre, and imaginary Z, Zim, components that originate mainly from the resistance and capacitance of the cell, respectively. A suitable equivalent circuit, shown in the inset of Figure 2B, was carefully selected to reflect the real electrochemical process and to enable fit producing accurate values. A modified Randles circuit consists of the ohmic resistance, RS, of the electrolyte solution, the electronic charge transfer resistance, RCT, in series with the finite length Warburg, W, and in parallel with a constant phase element, CPE, associated with the double layer and reflects the interface between the assembled film and the electrolyte solution. The solution resistance, RS, is the resistance between the aptamer-modified electrode and the reference electrode. The high frequency semicircle of the Nyquist diagram corresponds to the charge transfer resistance, RCT, in parallel with the CPE. The former represents the electron transfer kinetics of the redox probe at the electrode surface, whereas the latter corresponds to a nonlinear capacitor accounting for the inhomogeneity of the formed film.17 The diameter of the semicircle corresponds to the interfacial resistance at the electrode surface, the value of which depends on the dielectric and insulating features of the surface layer. On the other hand, the Warburg impedance, ZW, accounts for a diffusion-limited electrochemical process, presumably due to molecular motions within the film caused by conducting ion penetration. The rationale behind this electrochemical approach is that the binding between the target bacteria and the respective aptamers will block the electron transfer from a solution-based redox probe to the electrode surface. This could be attributed to the steric hindrance imposed by the bound bulky bacterial cells.18 Also, the repulsion between the negatively charged bacterial cell surface and the redox probe anions, [Fe(CN)6]3−/4−, might have contributed to the increased interfacial resistance observed upon bacterial binding to their respective aptamers. 19 Consequently, RCT will become increasingly high and can be used to monitor the binding event. It was observed that the RCT value increases linearly with increasing the concentration of S. typhimurium, in the range from 1 × 103 to 1 × 105 CFU mL−1, with the regression equation of y = 1376.3x + 3966.8 (R2 = 0.971), where y is the RCT value in Ω and x is the log concentration of S. typhimurium in CFU mL−1, as shown in the inset of Figure 2B and Table S2, Supporting Information. The relative standard deviation (RSD) values were between 0.3 and 2.1%. Beyond the upper bacterial concentration, the response became nonlinear, indicating the saturation of the surface with bacteria. The limit of detection (LOD) was 600 CFU mL−1, estimated from 3(Sb/m), where Sb is the standard deviation of the measurement signal for the blank and m is the slope of the analytical curve in the linear region.20 The sensor’s ability to distinguish between S. typhimurium and other Salmonella species, namely S. enteritidis and S.
Figure 3. (A) Nyquist plot (−Zim vs Zre) of impedance spectra for selectivity experiments performed using (a) buffer alone, (b) 5.1 mg mL−1 HSA, (c) 1 × 105 CFU mL−1 of S. enteritidis, (d) 1 × 105 CFU mL−1 of S. choleraesuis, and (e) 1 × 105 CFU mL−1 of live S. typhimurium. (B) Nyquist plot of impedance spectra for selectivity experiments performed using (a) buffer alone, (b) heat-killed 1 × 105 CFU mL−1 of S. typhimurium heated at 95 °C for 30 min, (c) 1 × 105 CFU mL−1 of S. typhimurium previously incubated with 1.0 μL of 20 mg mL−1 proteinase K, (d) 20 mg mL−1 proteinase K alone, and (e) 1 × 105 CFU mL−1 of live S. typhimurium.
Subsequently, it was utilized to develop the aptamer-based viability impedimetric sensor for bacteria (AptaVISens-B), as schematically depicted in Figure 1. The electrochemical characteristics of the developed aptasensor were investigated by cyclic voltammetry (CV), as shown in Figure 2A. The pretreated electrode surface presents a quasi-reversible voltammogram indicating that the redox reactions easily occurred on the bare gold surface, evidenced by very large redox currents (curve a). Formation of selfassembled monolayer (SAM) of the thiolated aptamer onto the electrode surface substantially reduced the electrode current, 8968
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choleraesuis, was confirmed by EIS experiments, as shown in Figure 3A. It was observed that incubation of the sensor with 1 × 105 CFU mL−1 of S. enteritidis caused a 27.5% increase in the RCT value (9103 Ω), whereas incubation with 1 × 105 CFU mL−1 of S. choleraesuis caused a 34.2% increase in the RCT value (9273 Ω). Percentages were obtained with reference to incubation with buffer alone (0%, 8406 Ω) and with 1 × 105 CFU mL−1 of live S. typhimurium (100%, 10940 Ω). The specificity of the sensor was also tested using 5.1 mg mL−1 human serum albumin (HSA, Sigma-Aldrich) which caused a 10.1% increase in the RCT value (8662 Ω). Furthermore, the ability of AptaVISens-B to distinguish between live and heat-killed S. typhimurium was also tested, as shown in Figure 3B. It was observed that incubation of the aptasensor with heat-killed bacteria (1 × 105 CFU mL−1 of S. typhimurium heated at 95 °C for 30 min) caused a 13.8% increase in the RCT value (6974 Ω). Incubation of the aptasensor with 1 × 105 CFU mL−1 of S. typhimurium previously incubated with 1.0 μL of 20 mg mL−1 proteinase K (Ambion, U.S.) for 30 min caused only 4.2% increase in the RCT value (6741 Ω), whereas incubation with 20 mg mL−1 proteinase K alone caused a 2.4% decrease in the RCT value (6582 Ω). Percentages were obtained with reference to incubation with buffer alone (0%, 6640 Ω) and with 1 × 105 CFU mL−1 of live S. typhimurium (100%, 9054 Ω). This work represents the proof of principle for the first aptamer-based viability sensor for bacteria (AptaVISens-B) taking advantage of the tunable specificity of aptamers, which enables the sensor to distinguish between live and killed bacteria. It also opens a new venue for the development of a variety of viability sensors for detection of many other microorganisms and spores. This is particularly important in sterility tests and for validating the efficiency of sterilization. Finally, aptamers either alone or coupled with antibiotics were recently reported as highly capable of inhibiting pathogenic bacteria such as Bacillus cereus.21 This trend is recently adopted to overcome the persistent problem of antibiotic resistance. The inhibitory effect of the developed aptamers on S. typhimurium is currently under investigation in our laboratories, and further results will be released in due course.
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work. The bacterial isolates for aptamer selection were kindly provided by the Federal State Institution of Health “Center for Hygiene and Epidemiology of the Krasnoyarsk Territory”.
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ASSOCIATED CONTENT
S Supporting Information *
Aptamer selection and cloning and sequencing procedures, flow cytometric analysis, preparation of the sensor, electrochemical measurement, DNA sequencing results of the selected aptamer pool, and equivalent circuit element values of the impedimetric sensor are presented in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: 613-562-5600 (1898). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Authors thank the Natural Sciences and Engineering Research Council of Canada, the Ministry of Education and Science of the Russian Federation (the Federal Target Program), and Siberian Branch of Russian Academy of Science for funding this 8969
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