Electrochemical sensing and fluorescence imaging of E . coli O 157 : H 7 based on aptamer-conjugated semiconducting nanoparticles

Aptamers are selective molecules against to various sizes of targets from small molecules to mammalian cells. Here, we reported QDs containing electrochemical aptasensor for the detection of E. coli O157:H7. The electrode surfaces were modified by cysteamine (CysN), which has amine and thiol groups, via self-assembled monolayer formation. The carboxyl-functionalized quantum dots (QD) and aptamers (Apt) were conjugated to cysteamine modified gold electrodes. Linear range for E. coli O157:H7 was from 1 to 10 2 CFU/mL after incubation on CysN/QD/Apt modified Au surfaces. QDs provide fluorescence surface, so that adhesion of cells was followed using fluorescence microscope. Adhered cells were also imaged by scanning electron microscopy. Finally, cell analysis was carried out in real samples.


Introduction
Aptamers (Apts) are synthetic nucleic acids and can be selected using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method.[1][2][3].Their unique properties against to targets make them useful for the fabrication of aptasensors.In different configuration such as electrochemical or optical aptasensors can be designed.Also they are selective against to various types of molecules from small compounds such as glucose [4], illicit drugs [5], bacteria [6] to mammalian cells such as cancer cells [7].Aptamer-based biosensors (aptasensors) are able to detect pathogens with high selectivity [8][9][10].Compared to antibody or enzyme based biosensors, aptasensors have many advantages such as higher selectivity, higher stability, affinity and lower cost [11][12][13][14].
Escherichia coli (E.coli) is a diverse group of bacteria.E. coli O157:H7 which is commonly identified pathogenic strain produces toxins that cause several diseases such as gastrointestinal problems and bloody diarrhea [15,16].E. coli O157:H7 has been found in food stuffs such as meat, fruit juice, milk and products, salami, lettuce [17].The detection, identification, and quantification of E. coli O157:H7 is critical in clinical diagnosis, disease control, environmental monitoring and food safety.
[20] and optical assays using organic dye molecules [21] are conventional identification methods for E. coli O157:H7.Although standard microbiological techniques allow the detection of bacteria, these processes are relatively slow and lack of high performance detectability and specificity analysis of target structure.Opposed to conventional assays, electrochemical sensing offers several benefits over traditional techniques such as short time, high-yield screening, simultaneous analysis, advanced detectability and unlabelled detection methods and devices [22].
We described here a novel electrochemical assay combining with a specific aptamer against to E. coli O157:H7 outer membrane proteins.Furthermore, carboxyl functionalized QDs were used together with aptamer to prepare aptasensors via EDC/NHS (1ethyl-3-(3-dimethylaminopropyl) carbodiimide/Nhydroxysuccinimide) chemistry [23] and the designed surfaces were applied to image and determine E. coli O157:H7.

Chemicals and reagents
Escherichia coli O157:H7 cells were retrieved from DSMZ (DSM 19206, German Collection of Microorganisms and Cell Cultures; Braunschweig, Germany).2-(N-morpholino) ethane sulfonic acid (MES) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) were purchased from Sigma and EDC/NHS was dissolved in MES Buffer, pH 6.0.Carboxyl Quantum Dot was from Invitrogen.Specific aptamer with amine groups against to E. coli O157: H7 was obtained from IDT and the sequence of aptamer is [24]; 5'-/5AmMC6/ATCCGTCACACCTGCTCTACGGCGCT CCCAACAGGCCTCTCCTTACGGCATATTATGG TGTTGGCTCCCGTAT-3' E. coli O157:H7 cells were cultivated in 50 mL Luria Bertani (LB) medium at 37 °C with 175 rpm [25].LB medium consists of tripton: 10 g/L; yeast extract: 5.0 g/L and NaCl: 10 g/L.During storage of cells for a month, they were seeded on agar; 15 g/L.To test the specificity of aptasensor, Gluconobacter oxydans (Gram (-) bacteria) were added to the aptamer modified surfaces instead of E. coli cells.G. oxydans cells was cultivated in 50 mL of growing medium.It consists of yeast extract (5.0 g/L) and glucose (5.0 g/L) (at 28 °C with 175 rpm) [26].Cultivated cells were used in late logaritmic phase.E. coli K12 cells were from Microbiology Department Culture Collection of Ege University, Faculty of Science and were cultivated in 50 mL LB medium.

Apparatus
The basic equipments used in the project are listed as incubator (New Brunswick Scientific, USA), autoclave (Hirayama, Japan), centrifuge (Experiment, England), pH meter (Han Instrument, USA).Electrochemical measurements were acquired from PalmSens electrochemical measurement unit.Gold electrodes were utilized as working electrode, a platinum electrode as counter electrode and Ag/AgCl as reference electrode during measurements.

Gold surface modification with the QD and aptamers
The pathway to modify gold surface with QD and aptamer is as follows: Firstly, 10 µL of 100 mM cysteamine (dissolved in 500 µL of Milli-Q water) was placed on Au electrode and incubated for 1 hour.Then, 5 µL of 0.5 M EDC, 5 µL of 0.125 M NHS, 12.5 µL of 2.5 µM Apt and 2,5 µL QD were retained on to the Au surface.The mixture was incubated for 2 hours.Furthermore, E. coli cells in different numbers were added and incubated for 45 minutes.In Fig. 1, the schematic representation of aptamer and QD modified Au surfaces was illustrated.To test the aptansensor success for the detection of E. coli in real samples, E. coli cells were spiked to tap water and dilutions of cells were carried out using the same tap water samples.The obtained signal after addition of tap water, including E. coli cells was used to calculate numbers of E. coli in samples using the linear graph.SEM images of modified surfaces were taken.The samples were dropped on to gold covered glass slides.The modified slides were placed onto copper grid surface.After drying samples, they were positioned onto container and imaged using the microscope.

Characterization of CysN/QD/Apt surfaces
As described in the method section, after the electrode modification with Apt and adhesion of the cells, cyclic voltammogram (CV) were formed in SPB (pH 7.0, 50 mM) containing 5.0 mM K3[Fe(CN)6] at 25 mV/sec between -0.4-0.5 V. K3[Fe(CN)6] is a probe for the following of electron transfer mechanism on the electrode surfaces.CV graphs showed an intense fall in the peak current when CysN, QD/Apt were covered and E. coli O157:H7 adhered on Au electrode due to the moderately limit for transfer properties of electrons (Fig. 2).The DPV peak current decreased also after the addition of E. coli O157:H7 cells (Fig. 3).This proves the successful modification of Au surfaces with aptamers and the adhesion of E. coli O157:H7 cells.The results of CV and DPV support each other.

The effect of the aptamer concentration on DPV signals
The CysN/QD/Apt modified Au surfaces were prepared using various concentrations of Apt.The graphic shown in Fig. 4 was obtained using different signals of the CysN/QD/Apt modified Au surfaces after addition of E. coli cells.As it is shown in Fig. 4, the most appropriate aptamer concentration was found to be 2.5 µM and the surfaces were prepared using aptamers in this concentration in all subsequent experiments.Concentrations of aptamer less than 2.5 mM used to prepare selective surfaces cause lower electrochemical signals.

Linear range for E. coli O157:H7
Altered numbers of E. coli O157:H7, from 10 to 10 2 CFU/mL, were prepared by serial dilution in % 0.9 NaCl solutions.A linear graph was obtained using the fall in the current depending on E. coli O157:H7 concentration within DPV measurements.The linear correlation was defined by the equation of y=-0.00435x+0.931(R 2 =0.998) and y shows ∆I (μA) and x shows the logarithm of the numbers of E. coli from 1 to 10 2 CFU/mL (Fig. 5).This linear range is suitable for E. coli O157:H7 detection in various samples and is compatible with the literature [27,28].
Fluorescence microscope images of CysN (Fig. 6A), CysN/QD/Apt covered (Fig. 6B) and adherent cells (Fig. 6C).As shown in Fig. 6C, the presence of E. coli cells on CysN/QD/Apt covered Au surface was imaged by fluorescence microscopy.The surface morphology of the CysN/QD/Apt covered Au electrode, before and after E. coli adhesion was imaged by scanning electron microscopy.After modification of Au surfaces with CysN, the obtained clear homogeneous surface morphology was shown in Fig. 7A.QD/Apt modified (in Fig. 7B) and the adhered cells on Au electrode were shown in Fig. 7C  and 7D.
And also selectivity of the aptasensor was confirmed using E. coli K12 and G. oxydans cells which are Gram (-) bacteria as a target instead of E. coli O157:H7 cells.As shown in Fig. 8, there is no interference effect of other cells.

Measurements on real samples
At the final step, the aptasensor was applied for the detection of E. coli cells in real samples.The spiked tap water samples were prepared by the addition of known numbers of E. coli cells with different dilutions.Using calibration graph for cells, the numbers of E. coli in tap water was calculated and the obtained results were compared with added numbers of E. coli.Added and found numbers of E. coli were 100 and 106.856±2.595CFU/mL, respectively.

Conclusion
Semiconducting nanoparticles are useful tool for electrochemical and optical applications.Use of them in fluorescence detection is one of the important areas because of their optical features.Here, E. coli O157:H7 was chosen as a model microorganism and electrochemical pathogen detection was carried out using aptamer modified gold electrodes.The analysis of the pathogen in tap water was also carried out without any interference which was included in matrix of sample.

Figure 1 .
Figure 1.Schematic representation of steps for modification of the gold electrode surfaces2.4.The electrochemical determination of the electrode responseCyclic voltammetry (CV) (between -0.4 V-0.4 V) and differential pulse voltammetry (DPV) (between -0.4 V and 0.5 V) techniques were performed after each modification in [Fe(CN)6] 3-/4-solution (dissolved in 50 mM pH 7.0 sodium phosphate buffer; SPB), as a redox probe (5.0 mM).E. coli adhesion onto the Apt modified gold electrode produced the decrease in the current signals which were related to the cell adhered onto the electrode.Changes in the sensor responses as a current were deliberated as follows; ∆I=Io-Ic (where Io is the current at without cell and Ic is the mean current in the presence of cells).To test the aptansensor success for the detection of E. coli in real samples, E. coli cells were spiked to tap water and dilutions of cells were carried out using the same tap water samples.The obtained signal after