DNA Detection of Toxoplasma gondii with a Magnetic Molecular Beacon Probe via CdTe@Ni Quantum Dots as Energy Donor

A new method for detection of Toxoplasma gondii via DNA sensing technology was developed in this study. It was based on the mechanism of fluorescence resonance energy transfer (FRET) in which multifunctional and magnetic-fluorescent CdTe@Ni quantum dots (mQDs) were utilized as energy donor and a commercial BHQ 2 as acceptor. The sensing probe was fabricated by labeling a stem-loop Toxoplasma gondiiDNA oligonucleotide with CdTe@Ni mQDs at the 5󸀠 end and BHQ 2 at 3󸀠 end, respectively. The surface assembly of CdTe on Ni core and the formation of CdTe@Ni were confirmed by XRD analysis. The sizes of CdTe, Ni nanoparticles, and CdTe@Ni were measured via TEM and XRD methods and estimated to be ∼3 nm, ∼15 nm, and ∼20 nm, respectively.The sensing ability was investigated by the fluorescence spectrum (FS). An obvious fluorescence recovery was observed when the complete complimentary target Toxoplasma gondii DNA was introduced, which did not happen in the case of the target DNA with one-base pair mismatch. Our research indicates that the current sensing probe is sensitive and specific in detection of Toxoplasma gondii DNA and has great potential in Toxoplasmosis diagnosis.


Introduction
In the past decades, one of the most prominent and fastest moving interfaces of nanotechnology was the synthesis and application of semiconductor nanomaterials (such as quantum dots (QDs)) in biological detection and bioimaging [1][2][3][4][5][6][7][8][9][10].The QDs have preeminent photophysical and chemical properties [11]: (1) efficient quantum yield, high brightness, and stability of the QDs against photobleaching; (2) the luminescence properties of QDs, which can be controlled by size due to the quantum confinement; (3) the luminescence spectra of QDs that reveal narrow emission bands and large Stokes shifts; (4) in contrast to organic fluorophores or transition metal complexes that exhibit broad absorbance bands and require specific wavelength excitation, QDs can be excited by a common high-energy excitation wavelength, leading to the size-controlled luminescence.Biological detection of fluorescent probes was based on fluorescence resonance energy transfer (FRET) mechanism [12], and QDs were used as an energy donor due to their excellent fluorescent properties.The molecular beacon sensing probe based on FRET was usually applied in DNA detection, in which the donor and acceptor were connected by a typical DNA strand with a stem-loop beacon structure.The FRET efficiency was controlled by altering the distance between the energy donor and acceptor [13].
Toxoplasma gondii is an obligate intracellular protozoan parasite belonging to the phylum Apicomplexa, subclass Coccidia.Toxoplasma gondii infections are known almost worldwide, and around one-third of the world's population is seropositive for this parasite.The infections of many Asians, including Chinese, were also reported [14].The efficient technology and early detection of Toxoplasma gondii or its shedding oocysts are necessary.Traditional method to detect the Toxoplasma gondii or diagnosis of Toxoplasmosis was time consuming and required professional knowledge.In current research, we developed a magnetic sensing probe for fast, convenient, and efficient analysis of Toxoplasma gondii DNA by using CdTe@Ni magnetic nanoparticles (MNPs) as an energy donor.Our data indicated that the target Toxoplasma gondii DNA could be successfully detected by the sensing probe.1.

Synthesis of CdTe Quantum Dots.
The CdTe was synthesized in aqueous phase according to the previous recipe described by Sarah et al. in 2011 [15] with some modifications.Briefly, CdCl 2 and MPA were mixed with mole ratio of 1 : 3 and adjusted the pH value to be 9.0.The prepared precursor NaHTe solution was added and refluxed for 5 h at 10 ∘ C.
By altering the refluxing time, QDs with different emission colors were prepared.
2.4.Preparation of CdTe@Ni Nanoparticles.Typically, CdTe@Ni NPs were prepared via the reduction method with hydrazine hydrate, as previously reported [16].Nickel chloride (NiCl 2 ⋅6H 2 O) and sodium hydroxide were added into ethylene glycol phase at 60 ∘ C for 0.5 h.Ni nanoparticles were magnetically precipitated and washed several times with ultrapure water and acetone.The resulting Ni nanoparticles were surfacely modified with 1,6-hexamethylenediamine (pH = 3.0), followed by adding CdTe QDs (mole ratio of Ni : CdTe was 1 : 9).The reactant solution was refluxed at 45 ∘ C for 0.5 h, and MNPs were achieved.

Fabrication of Molecular Beacon (MB) Probe.
The MB probe was obtained by reacting with 0.5 mL the as-prepared CdTe@Ni solution, 20 L probe DNA, 0.5 mL EDC, and 980 L phosphate buffers ((PBS), pH = 8.2) at ambient temperature for 24 h.
2.6.Detection of Target DNA. 100 L target DNA and 2.0 mL sensing probe DNA were added into a 5 mL penicillin bottle which contained 0.5 mL of 20 mM Tris-HCl buffer.The hybridization reaction happened at 37 ∘ C for 2 h.

Results and Discussion
3.1.Characterizations of CdTe QDs.The prepared CdTe QDs expressed excellent optical properties such as strong fluorescence intensity (FI), narrow emission peak, and broad UV absorption.Figures 1(a) and 1(b) were the fluorescence spectrum (FS) and UV-vis absorption spectrum, respectively.The emission colors varied from blue to red with the increase of emission wavelength (Figure 1(a)).Therefore, CdTe QDs refluxed for 4 h was used throughout the experiment due to its good fluorescent properties.The size of the synthesized CdTe was calculated using the polynomial fitting function [17]: where the  is the absorption maxima of the QDs.And the diameter of CdTe (4 h) was 2.6 nm.The concentration of CdTe QDs solution was 1.29 × 10 −6 M, determined via Lambert-Beer's law [18].The corresponding fluorescence quantum yields (Φ  ) were estimated according to [19]: where  and   were the absorbance of the sample and standard at the excitation wavelength, respectively. and   were the areas under the fluorescence curves of the QDs and standard, respectively. and   are the refractive indices of the solvent used for the sample and standard, respectively.Rhodamine 6 G in ethanol (Φ  = 0.95) was used as the standard [16].Then, the calculated value of Φ  was 27.3%.CdTe QDs were further characterized by transmission electron microscopy (TEM) (shown in Figure 2).TEM data indicated that the CdTe QDs had a favorable dispersity, and the size was ca.3.0 nm according to theoretical calculated value.

Characterizations of CdTe@Ni
NPs.The prepared Ni magnetic nanoparticles (MNPs) were characterized by X-ray diffraction (XRD) (Figure 3) and TEM (Figure 4(a)).The size of Ni MNPs was determined to be 10.5 nm, based on XRD analysis and the Scherrer equation [19].The average size of Ni MNPs analyzed by TEM was 13.0 nm, and obvious agglomeration phenomenon of Ni MNPs was observed (Figure 4(a)).However, after the Ni MNPs was further modified by 1,6hexamethylenediamine, the modified Ni MNPs had a better dispersity than before, and the average size was about 20.0 nm (Figure 4(b)).It confirmed that 1,6-hexamethylenediamine played a crucial role in reducing agglomeration.
The modified MNPs were further characterized by Zeta potential analysis.The Zeta potential value was 5.67 as shown in Figure 5, which means that particle surface was electropositive.This result indicated the successful surface modification of 1,6-hexamethylenediamine, which was good for CdTe@Ni synthesis.
The prepared CdTe@Ni was characterized with TEM (Figure 6) and XRD (Figure 7).The diameter of CdTe@Ni was uniform and estimated to be ca.35.0 nm (Figure 6).The increase of 20 nm in diameter was observed compared with the Ni (15 nm), which indicates that the CdTe was successfully capped on the Ni core.Little agglomeration was observed   due to the magnetism of CdTe@Ni.Moreover, the greenish CdTe@Ni was suspended well in aqueous solution and conveniently precipitated by a common magnet (Figure 6, inner graph).XRD patterns in Figure 7 indicate that the intensity of NiO (111) and Ni (111) was obviously decreased after formation of CdTe shell on the Ni particle core, which indicates that CdTe@Ni QDs was successfully achieved.

Sensitivity and Specificity of Prepared MB
Probe.About 56% of CdTe@Ni FI was decreased by comparing peak 1 and peak 3 as shown in Figure 8(a), which indicated that the MB probe was successfully created when using CdTe@Ni as energy donor and BHQ 2 as acceptor.However, the data in Figure 2(a) peak 3 showed low FI of the MB probe (commonly named as background noise) since the expiated energy from CdTe@Ni donor was not completely quenched by the BHQ 2 .It may involve the following possibilities.Firstly, the fluorescence emission spectra of CdTe@Ni donor and the UV absorption spectrum of BHQ 2 were not perfectly overlapped after formation of the core-shell structure (which usually causes the size change of donor), and the fluorescence emitted by the donor was not completely absorbed, thus showing a certain degree of fluorescence.Secondly, compared with the small organic molecules BHQ 2 , CdTe@Ni with greater surface area had more trap states, resulting in the emission energy not totally going to be absorbed by the acceptor.The FI was thoroughly recovered after adding the complementary target DNA, which revealed that the DNA sensing system had excellent sensitivity.
A mathematic modeling and practical method were proposed in our later research to reveal the possible factors that caused and decreased the noise, and the data will be reported elsewhere.Currently, the MB solution was diluted 4 times to decrease the effect of background noise for investigating the specificity.The obvious differences in the fluorescence intensity recovery (FR) were confirmed when different target DNA was applied in MB sensing probe solution.Compared with the complementary DNA (Figure 8(b), peak 5), the one-base mismatched DNA (Figure 8(b), peak 2 to peak 4) resulted in a much lower FR.This clearly demonstrates the excellent specificity of molecular beacons in detecting Toxoplasma gondii DNA.The difference in the MB hybridization between perfectly matched and single-base mismatched DNA relies on the stability of the newly formed DNA duplexes.Length of the hybridization sequence, location of mismatch bases in the sequence, and hybridization temperature have effects upon duplex stability.Therefore, high specificity with onebase mismatch DNA identification capability can be obtained by using carefully designed molecular beacon probes and optimized hybridization conditions.
FI was gradually reduced with the time-elapsing, and 30% of FI disappeared after 15 days of storage.The red-shift of emission peak was observed simultaneously.However, longterm stability is expected for the further applications, and the MB probe can be stabilized at 4 ∘ C for one month under the light-shielding condition.The MB limit of detection (LOD) was investigated via calibration curve (Figure 9) and calculated to be 2.70 × 10 −9 mol/L.

Conclusion
The CdTe@Ni nanoparticles with magnetic and fluorescent properties were synthesized and successfully used in molecular beacon (MB) probes as energy donors in Toxoplasma gondii DNA detection.The resulting data demonstrated that the sensing probe was sensitive, rapid, and specific in detecting the Toxoplasma gondii target DNA.This study indicates that the sensing probe has great potentials in early detection of Toxoplasmosis.

Figure 2 :
Figure 2: TEM image of the prepared CdTe QDs.

Figure 3 :
Figure 3: XRD pattern and selected area electron diffraction (inner graph) of Ni MNPs.

Figure 8 :Figure 9 :
Figure 8: (a) The FS of molecular beacon sensing system.Target DNA; line 1: CdTe@Ni solution, line 2: MB solution after adding target DNA, and line 3: MB solution.(b) The FS of FRET after adding various target DNA; line 1: MB solution without target DNA, line 2 to line 4: adding target DNA with one mismatch, and line 5: adding completely complementary target DNA of Toxoplasma gondii.

Table 1 :
The DNA sequences in experiment.