Paramagnetic Particles Isolation of Influenza Oligonucleotide Labelled with CdS QDs
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- Krejcova, L., Hynek, D., Kopel, P. et al. Chromatographia (2013) 76: 355. doi:10.1007/s10337-012-2327-0
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In this study, we describe hybridization design probes consisting of paramagnetic particles and quantum dots (QDs) with targeted DNA, and their application for detection of avian influenza virus (H5N1). Optical properties of QDs were beneficial, but the main attention was paid to the electroactivity of metal part of QDs and ODNs themselves. Differential pulse voltammetry was used for detection of cadmium(II) ions and square wave voltammetry for detection of cytosine–adenine peak in ODN-SH-Cd complex. It clearly follows from the obtained results that the optimized conditions were temperature of hybridization 25 °C, time of hybridization 35 min, and concentration of ODN-SH-Cd complex 20 μg mL−1. The detection limit (3 signal/noise) was estimated as 15 ng mL−1 of ODN-SH-Cd.
KeywordsBiosensorsVoltammetryAutomated separationNanoparticlesQuantum dotsHybridizationVirus
Influenza is an infectious disease caused by RNA viruses of the family Orthomyxoviridae. Influenza viruses can be found in the aerosols formed by sneezing and coughing and may cause acute infection of upper respiratory tract [1, 2]. Vaccine against influenza exists; however, it is effective for one year against selected subtypes. This is due to mutational changes in the structure of the virus, thus the reuse of the same vaccine in the following year does not have a protected effect [3, 4]. It is clear that flu viruses are very susceptible to change antigenic equipment by drift and shift. These changes can easily create a new subtype, as in the case of highly pathogenic avian influenza. Owing to this, a great amount of research is being carried out in the search for rapid and more reliable detection methods [1, 5, 6].
Nucleic acid hybridization on solid bases is widely used in biotechnology for the isolation of targeted DNA. Among numerous methods used in this field, many probe–target DNA assays use oligo-conjugated paramagnetic particles (MPs) for the isolation of nucleic acids of interest [7, 8]. The advantage of magnetic separation is in the possibility of modifying the surface of MPs, and thus the elimination of interfering adsorption of non-target biomolecules [9–11], because MPs are able to respond to external magnetic field, which is used for efficient separation. Both magnetic separation and modification of MPs surfaces are beneficial for DNA isolation [12–14].
Preparation of CdS QDs
Preparation of CdS Labelled H5N1 Influenza Oligonucleotide (ODN-SH-Cd)
ODN-SH H5N1 5′-SH-CTTCTTCTCTCTCCTTGAGG-3′ (100 μL, 100 μg mL−1) was mixed with a solution of CdS QDs (100 μL). This mixture was shaken for 24 h at room temperature using Vortex Genie2 (Scientific Industries, New York, USA). Subsequently, solution was dialysed against 2,000 mL of mili Q water (24 h, 4 °C) on Millipore membrane filter 0.025 μm VSWP. During dialysis, the sample was diluted to 800 μL. Diluted sample was concentrated to 500 μL final volume on a centrifugal filter device Amicon Ultra 3k (Millipore, Billerica, USA). Centrifuge 5417R (Eppendorf, Hamburg, Germany) was utilized with following parameters 15 min, 4,500 rpm, 15 °C.
Robotic Pipetting Station
For automated samples handling before their electrochemical analysis, an automated pipetting station ep-Motion 5075 (Eppendorf) with computer controlling was used. Positions C1 and C4 were thermostated (Epthermoadapter PCR96). In B1 position, module reservoir for washing solutions and waste were placed. Tips were placed in positions A4 (epTips 50), A3 (epTips 300) and A2 (epTips 1000). Transfer was ensured by a robotic arm with pipetting adaptors (TS50, TS300, TS1000) numeric labelling refers to maximal pipetting volume in μL and a gripper for platforms transport (TG-T). The program sequence was edited and the station was controlled in pEditor 4.0. For sample preparation, two platforms were used: Thermorack for 24 × 1.5–2 mL microtubes (position C3), which was used for storage of working solutions, 96-well microplate with well volume of 200 μL (position C1), which was thermostated. After the separation, the MPs were forced using Promega magnetic pad (Promega, Madison, USA) (position B4) and the solutions were transferred to a new microplate.
Automatic Isolation of ODN-SH-Cd
Automatic pipetting station ep-Motion 5075 (Eppendorf) with original devices was used for fully automated influenza-derived oligonucleotide isolation process. The buffers used in isolation part of experiment were as follows: phosphate buffer I: 0.1 M NaCl + 0.05 M Na2HPO4 + 0.05 M NaH2PO4; phosphate buffer II: 0.2 M NaCl + 0.1 M Na2HPO4 + 0.1 M NaH2PO4; and hybridization solution: 100 mM Na2HPO4 + 100 mM NaH2PO4, 0.5 M NaCl, 0.6 M guanidium thiocyanate, 0.15 M Trizma base adjusted by HCl on pH of 7.5.
The protocol was as follows: 10 μL of Dynabeads Oligo (dT)25 (1 μm diameter, Hämeenlinna, Finland) was dispensed in each well in the plate (PCR 96, Eppendorf. Plate was subsequently transferred to the magnet, stored solution from nanoparticles was aspirated to waste, and beads were further washed three times with 20 μL of phosphate buffer I. The next step was first hybridization. 10 μL of polyA-modified anti-sense H5N1 oligonucleotide and 10 μL of hybridization buffer (0.1 M phosphate buffer, 0.6 M guanidium thiocyanate, 0.15 M Tris) were added into each well. Further, the plate was incubated (15 min, 25 °C, pipetting), followed by three times washing with 20 μL phosphate buffer I. The next step was second hybridization. 10 μL of Cd labelled H5N1 oligonucleotide and 10 μL of hybridization buffer (0.1 M phosphate buffer, 0.6 M guanidium thiocyanate, 0.15 M Tris) were added to each well, and the plate was incubated (15 min, 25 °C, pipetting), followed by three times washing with 20 μL of phosphate buffer I. Afterwards, 30 μL of elution solution (phosphate buffer II) was added into each well, and the plate was incubated (5 min, 85 °C, pipetting). After elution step, the plate was transferred to the magnet, and product from each well was transferred to separate well. The whole procedure was optimized by other authors [28–30].
Methods for Detection of CA and Cd Peak (ODN-SH-Cd)
Measurements were performed at 663 VA Stand, 800 Dosino and 846 Dosing Interface (Metrohm, Zofingen, Switzerland) using a standard cell with three electrodes. A hanging mercury drop electrode with a drop area of 0.4 mm2 was employed as the working electrode. An Ag/AgCl/3 M KCl electrode served as the reference electrode, while the auxiliary electrode was a glassy carbon electrode. All measurements were performed in the presence of 0.2 M acetate buffer (0.2 M CH3COOH + 0.2 M CH3COONa, pH 5.0) at 25 °C. Samples were deoxygenated by argon (99.99 %, 120 s). For smoothing and baseline correction, the software GPES 4.9 supplied by EcoChemie (Utrecht, Netherlands) was employed. For detection of DNA, CA peak measured by square wave voltammetry (SWV) was used. The parameters of electrochemical determination were as follows: initial potential 0 V; end potential −1.85 V; frequency 10 Hz; potential step 0.005 V; and amplitude 0.025 V. For electrochemical detection of cadmium (Cd peak), differential pulse voltammetry (DPV) was used. The parameters of electrochemical determination were as follows: initial potential −0.9 V; end potential −0.45 V; deposition potential −0.9 V; duration 240 s; equilibration time 5 s; modulation time 0.06; time interval 0.2 s; potential step 0.002 V; and modulation amplitude 0.025.
Scanning Electron Microscope
A modern scanning electron microscope (SEM) with motorized stage, full software control and image acquisition was recognized as a relatively easy way for automated high-resolution documentation of particles. For each experiment, three independent samples of particles on different tablet sections (glass, pure Si, and Millipore syringe filters) were documented. FEG-SEM TESCAN MIRA 3 XMU (Brno, Czech Republic) was used for documentation. This model is equipped with a high brightness Schottky field emitter for low noise imaging at fast scanning rates. The SEM was fitted with Everhart–Thronley type of SE detector, high speed YAG scintillator-based BSE detector and panchromatic CL Detector.
Data were processed using MICROSOFT EXCELs (USA) and STATISTICA.CZ Version 8.0 (Prague, Czech Republic). The results are expressed as mean ± SD unless noted otherwise. The detection limits (3 signal/noise, S/N) were calculated according to Long and Winefordner , whereas N was expressed as standard deviation of noise determined in the signal domain unless stated otherwise.
Results and Discussion
Rapid detection of the presence of virus represents a challenge for modern bioanalytical chemistry. For this purpose, MPs bring many advantages including possibility of miniaturization of the instrument as lab-on-chip . Moreover, paramagnetic particles are suitable for sensing, therapeutic and diagnostic purposes . In this study, particles modified with short thymine chain (dT25) were used (Fig. 2a). Next step was labelling of influenza derived thiolated oligonucleotide with CdS QDs. These QDs were prepared from cadmium nitrate tetrahydrate according to procedure mentioned in the “Experimental” section. The prepared CdS QDs had fluorescent properties as shown in Fig. 2b. The possibilities of labelling of nucleic acids and proteins with QDs are discussed by Joseph Wang and Cliphord Mirkin and showed many advantages for potential applications in nanomedicine [33–37]. The last part of preparation of sensing assay was full automation of the isolation with subsequent electrochemical detection of CdS labelled H5N1 influenza derived oligonucleotide (ODN-SH-Cd). Scheme of ODN-SH-Cd isolation and electrochemical detection is shown in Fig. 1.
Determination of Cadmium(II) Ions and ODNs
After characterization of labelled and non-labelled ODNs, the optimization of accumulation times for both ODNs was done. Binding of CdS to ODN-SH decreased CA peak in the whole tested interval, but in both cases, the value of 120 s was selected as optimal for further studies (Fig. 4b). Dependences of CA peak height on both labelled and non-labelled ODN concentrations are shown in Fig. 4c. It is shown that the both dependences have polynomial calibration equations. For ODN-SH, the equation was y = −27.56x2 + 105.2x + 0.3250; R2 = 0.9984, n = 5, RSD = 3.2 and for ODN-SH-Cd y = −13.75x2 + 59.57x − 0.8282; R2 = 0.9963, n = 5, RSD = 2.9. The detection limits (3 S/N) were estimated as 15 ng mL−1 of ODN-SH-Cd and 30 ng mL−1 of ODN-SH.
Optimization of Separation Process: Hybridizations
Paramagnetic particles (MPs) are able to respond to external magnetic field, which is used for efficient separation of different analytes from liquid samples. Isolation of biomolecules using MPs is usually followed by electrochemical detection. This way of isolation and detection is less time-consuming and it is highly sensitive to even small quantities of sample [28, 41]. In order to study using MPs as a tool for hybridization assays we designed a MP- and QD-based hybridization assay for the detection of avian influenza H5N1, respectively H5N1-derived oligonucleotide. Figure 1 shows a schematic view of the hybridization procedure for isolation and detection of target (ODN-SH-Cd). The separation procedure contains from four items arranged in the following order: (i) polyA-modified oligo probes (oligo anti-sense) bind on the surface of polyT MPs due to specific TA capturing, (ii) Cd QDs labelling target oligonucleotide, (iii) capturing of target QDs labelled oligonucleotide derived from an influenza sequence, and (iv) electrochemical detection of metal part of QDs marker by DPV. Electrochemical detection of influenza H5N1-derived oligonucleotide using SWV was connected too.
The third changing condition of hybridization process was concentration of ODN-SH-Cd complex. The concentration was changed within the interval from 2.5 to 20 μg mL−1 and both CA and Cd peaks were determined (Fig. 6b). As for previous parameters, the concentration of ODN-SH-Cd complex influenced measured peaks in the same way. Response of Cd peak increased linearly as it follows y = 2.202x + 56.61; R2 = 0.9958, n = 3, RSD = 3.8, and ODN peak quadratically as it follows y = −0.2013x2 + 7.412x + 32.04; R2 = 0.9838, n = 3, RSD = 5.6 with the increasing concentration of ODN-SH-Cd complex. It clearly follows from the obtained results that the optimized conditions were temperature of hybridization 25 °C, time of hybridization 35 min and concentration of ODN-SH-Cd complex 20 μg mL−1. Under these conditions, we can obtain the best efficiency in isolation and detection of influenza oligonucleotide.
The obtained results show that suggested method of isolation and detection of Cd labelled influenza-derived oligonucleotide is functional and provide electrochemically measurable signal of target molecule. Cd peak gives us the information about successful hybridization process, and ODN peak relates with the presence of ODNs in solution after hybridization process. The number of molecules determined from CA peak is approximately ten times higher than the number from Cd peak. This effect might be explained from breakage of creating complex after second hybridization in the other place that we assumed. Based on our previously published articles, the breakage of complex can proceed at A-T place [28–30]. We assumed that this situation can be explained from binding of more oligonucleotides to one CdS QD particle. This reasoning is in accordance with studies of Nolan et al.  and Ho et al.  who reported the use of QDs labelling oligo probes for hybridization with target DNA. Yet another published study is based on DNA-cross-linked CdS nanoparticles array  and shows similar effect.
Nowadays, research is directed towards finding methods for simultaneous detection of multiple DNA targets. The electrochemical coding technology is thus expected to open up new opportunities for DNA diagnostics [17, 45]. In this study, an optimized method for automated isolation of Cd-labelled influenza oligonucleotide using automated pipetting station has been proposed. The effects of hybridization temperature, hybridization time, and concentration of ODN-SH-Cd complex on CA and Cd peak height, which were determined electrochemically using SWV and DPV, were also demonstrated. The system proposed in this study can be used as electroanalytical tool for rapid detection of target oligonucleotide based on isolation by probe conjugated MPs.
Financial support from the projects NANIMEL GA CR 102/08/1546 and CEITEC CZ.1.05/1.1.00/02.0068 is gratefully acknowledged.