Biosensors and Bioelectronics 54 (2014) 195–198

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Short communication

Aptamer cocktails: Enhancement of sensing signals compared to single use of aptamers for detection of bacteria Yeon Seok Kim a,1, Jinyang Chung a, Min Young Song a,b, Jongsoo Jurng a, Byoung Chan Kim a,n a b

Center for Environment, Health and Welfare Research, Korea Institute of Science and Technology, Seoul 136-701, Republic of Korea Graduate School of Energy and Environmental System Engineering, University of Seoul, Seoul 130-743, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 7 August 2013 Received in revised form 16 October 2013 Accepted 2 November 2013 Available online 12 November 2013

Microbial cells have many binding moieties on their surface for binding to their specific bioreceptors. The whole-cell SELEX process enables the isolation of various aptamers that can bind to different components on the cell surface such as proteins, polysaccharides, or flagella with high affinity and specificity. Here, we examine the binding capacity of an aptamer mixture (aptamer cocktail) composed of various combinations of 3 different DNA aptamers isolated from Escherichia coli and compare it with one of the single aptamers using fluorescence-tagged aptamers. The aptamer mixtures showed higher fluorescence signal than did any single aptamer used, which suggests that use of aptamer mixtures can enhance the sensitivity of detection of microbial cells. To further evaluate this effect, the signal enhancement and improvement of sensitivity provided by combinatorial use of aptamers were examined in an electrochemical detection system. With regard to current decreases, the aptamer cocktail immobilized on gold electrodes performed better than a single aptamer immobilized on gold electrodes did. Consequently, the detection limit achieved using the aptamers individually was approximately 18 times that when the 3 aptamers were used in combination. These results support the use of aptamer cocktails for detection of complex targets such as E. coli with enhanced sensitivity. & 2013 Elsevier B.V. All rights reserved.

Keywords: Cell-SELEX Aptamer cocktails Bacteria Detection Signal enhancement

1. Introduction Conspicuous and rapid targeting of specific pathogens or their subpopulations in complex environments are regarded as the greatest challenge in the field of microbial diagnosis. Ligands having high affinity and specificity make diagnosis precise and efficient (Eaton et al., 1995). Antibodies and their variants are widely used as ligands, and their capabilities have been demonstrated in many applications in biotechnology, diagnostics, and disease treatment; however, preparation and modification of these antibodies are challenging and laborious tasks (Mayer et al., 2010; Zichi et al., 2008). Nucleic acid-based aptamers have been verified to be useful for generating high-affinity ligands for an array of targets ranging from small molecules to cells by SELEX (systematic evolution of ligands by exponential amplification) (Ellington and Szostak, 1990, 1992; Sefah et al., 2010; Tuerk and Gold, 1990). In contrast to antibodies, nucleic acid-based aptamers are relatively rigid because the backbone of nucleic acids has less torsional n

Corresponding author. Tel.: þ 82 2 958 5877; fax: þ82 2 958 5805. E-mail address: [email protected] (B.C. Kim). 1 Present address: Future Technology R&D Division, SK telecom, Sungnam, Gyunggi 463-784, Republic of Korea. 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.11.003

conformations than that of proteins, where the side chains have various degrees of torsional freedom; therefore, nucleic acid-based aptamers are tolerant to harsh environmental conditions (Eaton et al., 1995). Aptamers can be isolated in vitro and do not require immunization, tissue culture, or purification from serum for mass production. After determination of the aptamer sequence, large quantities of the specific oligomer can be synthesized by chemical or enzymatic processes at low cost and high efficiency (Graham and Zarbl, 2012). Cell-SELEX is a recent application of SELEX, in which representative cellular ligands (aptamers) are prepared against the surface of cells by repeated binding and amplification of ssDNA or RNA to living cells (Sefah et al., 2010). Cell-SELEX offers flexibility, because it can be applied to various cell types, from bacterial cells to cancer cells, to discover unknown biomarkers exposed on the surface of the cells (Kim et al., 2013; Sefah et al., 2010). In particular, bacterial cell-SELEX can be operated in nonadherent cell cultures to identify specific aptamers, and many components of the cellular surface such as polysaccharides, surface proteins, or flagella can be targets. The aptamers isolated against the bacterial cellular surface can be manipulated to construct bacterial diagnostic sensors that can be operated in a non-invasive and label-free manner without requiring cell lysis

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(Labib et al., 2012; Ohk et al., 2010). In our previous study (Kim et al., 2013), using bacterial cell-SELEX, we isolated several aptamers with high affinity and specificity owing to the various possible targets on the surface of cellular membrane; however, exact targets of these aptamers on Escherichia coli cells remain to be identified and verified. Considering the complexity of the cellular surface, we suspect that all aptamers do not share the same targets. Herein, we investigate the advantage of the combination of aptamers (aptamer cocktail) targeting the same bacterium to enhance the sensing signal, thereby enhancing the sensitivity of detection, over using a single aptamer as the sensing ligand in a biosensor. We performed a binding assay for single aptamers and aptamer cocktails to compare signal generation when the aptamers bound to the bacterium E. coli. Further, aptamer cocktails were immobilized on a screen-printed gold electrode for electrochemical detection of E. coli and their sensitivity and limit of detection were compared that of a single immobilized aptamer. These results indicate that aptamer cocktails, not only single aptamers, can be used to identify useful ligands for the construction of biosensors with enhanced sensitivity to detect complex targets that have many possible binding moieties for the ligands.

2. Materials and methods 2.1. Bacterial strains and culture conditions E. coli (KCTC 2571) was used as the target strain and grown in nutrient broth medium. Klebsiella pneumoniae (KCTC 2208), Citrobacter freundii (KCTC 2006), Enterobacter aerogenes (KCTC 2190), and Staphylococcus epidermidis (KCTC 1917) were used for specificity testing and were grown in nutrient broth. All strains were cultured at 37 1C, except C. freundii and E. aerogenes, which were cultured at 30 1C. All bacterial strains were obtained from the Korean Collection for Type Culture (KCTC). 2.2. ssDNA aptamers Three different ssDNA aptamers specific to E. coli, which were isolated in a previous study, were used in this study (Kim et al., 2013). The sequence of each aptamer was as follows: E1, 5′-GCA ATG GTA CGG TAC TTC CAC TTA GGT CGA GGT TAG TTT GTC TTG CTG GCG CAT CCA CTG AGC GCA AAA GTG CAC GCT ACT TTG CTA A-3′; E2, 5′-GCA ATG GTA CG G TAC TTC CCC ATG AGT GTT GTG AAA TGT TGG GAC ACT AGG TGG CAT AGA GCC GCA AAA GTG CAC GCT ACT TTG CTA A-3′; E10, 5′-GCA ATG GTA CGG TAC TTC CGT TGC A CT GTG CGG CCG AGC TGC CCC CTG GTT TGT GAA TAC CCT GGG CAA AAG TGC ACG CTA CTT TGC TAA-3′. All aptamers were custom-synthesized by Genotech Inc. (Daejeon, Korea), and all aptamers were dissolved into a concentration of 10 μM in distilled water for further use. 2.3. Fluorescence analysis for aptamer binding E. coli (KCTC 2571) were cultured in nutrient broth to the middle growth phase (108 CFU/ml) and centrifuged at 13,000 g for 10 min to remove the media. Subsequently, cells were washed 3 times in PBS (pH 7.4). FAM-labeled ssDNA aptamers were prepared as single aptamers (E1, E2, and E10) or aptamer cocktails (E1þ E2, E1 þE10, E2 þE10, and E1 þE2 þ E10) with various concentrations (0, 10, 25, 50, 100, 250, and 500 nM) in PBS. The molar ratio of each aptamer in the cocktails was the same. Then, 100 μl of the cell suspension (107 cells) was incubated with 100 μl of aptamer solution for 45 min at 25 1C with mild shaking. Cells were washed 2 times to remove unbound aptamers from cells by

centrifugation (13,000 rpm for 10 min) and were resuspended in PBS. Finally, the fluorescence intensity of each sample was measured using a fluorospectrophotometer (LS50B, PerkinElmer Co., USA). 2.4. Fluorescence imaging of E. coli with QD-labeled DNA aptamers To observe the binding of different aptamers on a single E. coli cell surface, the aptamers were labeled with 3 different quantum dots (QDs). Two QDs (NSQDs-AC530 and NSQDs-AC580, Nanosquare Inc.) were conjugated using the E1 and E2 aptamers, respectively. Another QD (Qdots655 ITK™ carboxyl, Invitrogen) was conjugated with the E10 aptamer. Carboxyl-functionalized QDs (5 μl, 8 μM) were incubated with 5 μl of 1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC) and Nhydroxysulfosuccinimide (Sulfo-NHS) (each 10 mM) for 2 h at 25 1C. The amine-modified aptamer (85 μl, 2 μM as the final concentration) in PBS buffer was added to the QD solution and incubated for 2 h at 25 1C with mild shaking. Finally, non-reacted COOH groups on QDs were blocked with ethanolamine (10 mM) for 2 h. Then, 3 QD/aptamer conjugate pairs were incubated with E. coli (107 cells) for 45 min at 25 1C. The cells were washed 2 times by centrifugation and resuspended in 40 μl of PBS buffer. The cell suspension was dropped onto a glass slide, and a cover slide was used to make a thin smear of the bacteria. Fluorescent images of the bacteria were observed under a fluorescence microscope (Olympus BX50). 2.5. Electrochemical detection of E. coli using aptamers immobilized on gold electrode chip Electrochemical analysis was performed at room temperature using an electrochemical analyzer CHI640D (CH Instrument, USA). The screen-printed electrodes composed of working (gold), reference (silver), and counter electrodes (gold). All electrodes were integrated in a single chip, and the working electrode had a diameter of 1.6 mm (DropSens, S.L., Spain). To fabricate the chip, each E. coli-specific DNA aptamer was first labeled with an amine group at 3′-end, and then immobilized as a single aptamer (E1, E2, or E10) or aptamer cocktails (E1þ E2, E1 þE10, E2 þE10, or E1 þE2 þE10). The electrodes were washed with 10 mM H2SO4 for 10 min before immobilization. To immobilize the aptamers on the electrode, the working electrode was treated with 10 mM 3,3′dithiodipropionic acid for 2 h to form a self-assembled monolayer (SAM) and then washed thoroughly with distilled water. The CVs of every electrode at this stage were measured for quality control and further aptamer modification after SAM modification (Fig. S1). Activation of carboxylic groups was performed on the electrode after incubation with 40 mM EDC and 40 mM sulfo-NHS for 1 h. Finally, 1 μM amine-modified DNA aptamer was incubated on an electrode for 1 h and washed thoroughly with distilled water. Then, ethanolamine (20 mM, pH 8.5) was applied to block the non-reacted COOH group for 1 h. Various concentrations of E. coli suspension (0–108 CFU/ml) in PBS were dropped onto the electrode and incubated for 1 h. After washing with distilled water, 70 μl of 5 mM K3Fe(CN)6 solution containing 100 mM KCl was dropped on electrode chip until all 3 electrodes are immersed. Then, cyclic voltametry (CV) was performed under electric potentials ranging from –0.2 to 0.4 V with a scan rate of 20 mV/s and a sample interval of 2 mV. To verify that the current change was caused by only specific interactions between E. coli and the aptamer, other bacteria species (K. pneumoniae, C. freundii, E. aerogenes, and S. epidermidis) were tested under identical conditions. Electrochemical data analysis was conducted, and the percent decrease in the current before and after the sample treatment (ΔI¼ (I0  I1)/I0  100) was

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measured. ΔI is the relative current change, and I0 and I1 represent the current before and after the sample treatment, respectively.

3. Results and discussion 3.1. Enhancement of fluorescence signal by using aptamer cocktails The whole-cell SELEX process has several advantages over the use of purified extracellular surface targets for the isolation of aptamers. Information regarding the extracellular surface targets is not necessary; moreover, aptamers can be isolated and selected against targets in their native conformation and physiological environment directly from the cellular surface without cell lysis. In addition, this method enables the isolation of a panel of aptamers that can bind to different moieties on the surface of the target cell (Cao et al., 2009; Cerchia et al., 2005; Pestourie et al., 2006). In our previous study, 3 E. coli-specific ssDNA aptamers having different affinities (E1: 12.4 nM, E2: 25.2 nM, and E10: 14.2 nM) were isolated, and we estimated that 3 ssDNA aptamers have different binding sites on the cell surface by a competitive binding assay (Kim et al., 2013). Therefore, we assumed that the usage of multiple aptamers (an aptamer cocktail) may improve the sensing performance for E. coli detection because 3 aptamers simultaneously can bind to different moieties on cell

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surface in a non-competitive manner (Fig. 1A). Initially, a fluorescence image of E. coli incubated with different QD-labeled aptamers was observed to confirm that the 3 different aptamers bound to a single E. coli cell surface simultaneously. A single E. coli cell emitted mixed wavelengths of green, orange, and red light, which indicates that the 3 different aptamers bound to the surface of a single E. coli simultaneously (Fig. S2). To determine whether this simultaneous binding of mixed aptamers could enhance the sensing signal, FAM-labeled aptamers were interacted with E. coli as a single aptamer or multiple aptamers (double and triple combinations), and the fluorescence intensity of the cells was analyzed. The total molar concentration of each combination and single aptamer tested was the same. Cell suspensions that were incubated with multiple aptamers showed higher fluorescence intensity than those incubated with single aptamers, possibly because of the cumulative effect of multiple aptamers (Fig. 1B). In particular, the fluorescence intensity of the triple aptamer cocktail was more than 100% higher than that of single aptamers. This result indicates that the usage of multiple aptamers can enhance the signal more efficiently than probing by a single aptamer for E. coli detection. This approach may be useful for not only bacterial cells but also for complex targets such as cancer cells or tissues that can support multiple binding moieties for specific receptor panels. However, this approach may not be effective for purified molecular targets such as proteins or small compounds because they bind to their target competitively. 3.2. Enhancement of electrochemical detection

Fig. 1. (A) Schematic concept of signal enhancement by aptamer cocktails for microbial cell detection. (B) Fluorescence intensity of cell suspensions obtained by the interaction with single aptamers or aptamer cocktails.

The effect of signal enhancement by the aptamer cocktail for E. coli detection was investigated in an electrochemical analysis. Single aptamers or multiple aptamers were immobilized on a screen-printed gold electrode chip using the same molar concentration. Then, various cell densities of E. coli were loaded on the electrode and the current change was evaluated. Fig. 2 shows the degree of current change according to the cell density for gold electrode chips immobilized with single aptamers or multiple aptamers. The current decrease at the same cell density was higher on the order of triple combination, double combinations, and single aptamers. We attributed this result to the different holding forces of aptamers to E. coli. When E. coli is loaded on an aptamer-immobilized electrode, it is captured by the aptamers and held on the electrode. However, E. coli is motile, and the interaction between the aptamer and E. coli is reversible; therefore, the holding force of aptamers to E. coli affects the electrochemical signal. Multiple aptamers immobilized on an electrode can capture E. coli more strongly than a single aptamer on the same electrode area (Fig. S3). Based on this result, the limit of detection was determined to verify the improvement of sensing performance by the aptamer cocktail. The limit of detection was calculated by adding the blank standard deviation multiplied by 3 to the blank signal (Medley et al., 2011). First, we calculated the LOD of ΔI using ΔIblank þ 3SDblank. Then, LOD of cell concentration was recalculated based on calibration curve obtained from correlation of cell concentration to ΔI. The LOD of the electrode immobilized with a single aptamer was in the range from 3.2  103 to 1.07  104 CFU/ml (average of single aptamer: 6778 CFU/ml), whereas an electrode immobilized with triple combinatorial aptamers produced an approximately 18-fold lower LOD value: 3.7  102 CFU/ml (Fig. 3). This finding supports the view that combinatorial use of aptamers for E. coli detection is more efficient than the use of single aptamers. The specificity of the electrode chip immobilized with the aptamer cocktail (triple combination) was examined for other bacterial species (K. pneumoniae, C. freundii, E. aerogenes, and S. epidermidis). Other bacterial species did not significantly reduce

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Fig. 2. (A) Current reduction depending on cell density produced by using single aptamers or aptamer cocktails immobilized on a gold electrode. The values for the current reduction of single aptamers or aptamer cocktails (double) are averaged from individual measurements of each set (E1, E2, and E10 for single aptamers and E1 þ E2, E2 þ E10, and E1 þ E10 for the aptamer cocktail (double)). The solid line is calibration curve obtained from correlation of cell concentration to current reduction change for calculation of LOD, (B) cyclic voltammetry diagrams demonstrate Escherichia coli detection by single aptamers (E2) or an aptamer cocktail (E1 þE2 þ E10) immobilized on the gold electrode.

is effective and superior to the conventional use of single aptamer. This approach may be used for other complex targets of aptamers such as other bacteria, cancer cells, and tissues.

Acknowledgment This work was financially supported by the Korea Institute of Science and Technology (KIST) Institutional Research Program (2E23952 and 2V02780).

Fig. 3. LOD level of E. coli detection performed by single or aptamer cocktails immobilized on an electrode chip.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.11.003.

the current compared to E. coli (Fig. S4). This finding also indicates that the specificity of E. coli detection was improved by use of the aptamer cocktail. The signal (current change in percentage) ratio of E. coli to other species was higher for the triple aptamer combination than for the single aptamer. Improvement of selectivity and reduction of false-positive results is another advantage of using multiple aptamers for bacterial cell detection (Beier and Gross, 2006; Bronner et al., 2004), probably because of increased signal or synergistic signal generation. 4. Conclusion In this study, we have demonstrated that the use of aptamer cocktails could effectively increase the sensitivity of detection methods and systems for both fluorescence assay and electrochemical detection of E. coli. As a potential application in electrochemical sensor, aptamer cocktail was successfully employed for the detection of E. coli with 18 fold lower detection limit compared to single aptamer. Due to the combination of different receptors to the same target on the electrode, aptamer cocktail showed better performance in detection signals than single aptamer. All these results support that the use of aptamer cocktail for cellular target

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