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Ratiometric sensor based on imprinted quantum dots-cationic dye nanohybrids for selective sensing of dsDNA
Analytical Biochemistry 591 (2020) 113540
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Ratiometric sensor based on imprinted quantum dots-cationic dye nanohybrids for selective sensing of dsDNA
T
Taner Arslan, Orhan Güney∗ Istanbul Technical University, Departments of Chemistry, 34469, Maslak, Istanbul, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Quantum dots Ratiometric sensor Surface imprinting dsDNA detection
A ratiometrically responsive sensor for dsDNA is reported, based on molecularly imprinted polymer coated quantum dots (MIP-QDs). A new platform is described for probing dsDNA by tracing the “turn on” fluorescence signal of malachite green (MG) as a cationic dye and “turn off-on” room temperature phosphorescence (RTP) signal of MIP-QDs/MG nanohybrids. The interaction between MIP-QDs surface and MG discloses an intense quenching in RTP (turning off) by a phosphorescence resonance energy transfer (PRET) process. After the addition of dsDNA, MG molecules escape from the MIP-QDs surface and intercalate into the dsDNA, resulting in the restoration of RTP intensity of MIP-QDs (turning on) and also enhancing in fluorescence of MG. This outcome hereby can be employed for the selective sensing of dsDNA via optical response. The ratio of fluorescence enhancement of MG to RTP intensity of MIP-QDs is proportional to the concentration of dsDNA in the range of 0.089–1.79 μg/mL with a detection limit (3σ/K) of 19.48 ng/mL under the optimized experimental conditions.
1. Introduction As the basic hereditary material in humans and almost all other organisms, DNA is crucial for the transportation of genetic information and the determinant of species continuation. Even slight change in structure of double-stranded DNA (dsDNA) may initiate the alteration of genetic characteristics and the occurrence of various diseases [1]. The level of DNA concentration in human plasma/serum might be sign of a variety of tumors and determination of DNA in urine could assist for early recognition of cancer, tuberculosis, HIV, malaria and potentially many other diseases [2]. Selective, sensitive and cost-effective detection of DNA has great significance in the diverse field including food safety, genetic diseases, pathology, pharmacokinetics, clinical diagnosis and treatments [3]. Quantitative detection of DNA has an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis. Currently, DNA diagnostic techniques are routinely used not only in research laboratories, but also in clinical and forensic practice. In the past decade, several strategies have been developed for DNA assay with including electrochemistry [4], surface-enhanced Raman spectroscopy (SERs) [5], colorimetric [6] and fluorescence methods [7]. Owing to the innate advantages, luminescence methods are fast, simple and high sensitive for DNA detection. Chromophoric dyes such as ethidium bromide, acridine orange and malachite green can form a
∗
stable fluorescent complex via intercalating with dsDNA [8–10]. Luminescence methods based on single emission could be faced by some interfering factors such as concentration of luminophore, output of instrument and specification of conditions. On the other hand, the ratiometric dual-emission can elude these effects and determine the analytes in complex samples through recording the ratios of emission intensities at two different wavelengths [11]. There are two main approaches for DNA analysis based on QDs; one way is “turn off” method (fluorescence quenching) and another way is “turn off-on” method (fluorescence quenching recovery) [10,12]. Analysis of DNA can be performed quickly by fluorescence “turn off” method, but false positive results may occur because non-specific disturbances caused by other quenchers in the medium instead of the analyte will result in a “turn off” state [13]. Furthermore, fluorescence “turn off-on” sensors have more selectivity than “turn off” methods [14]. Therefore, fluorescent “off-on” sensors provide more dependable detection performance and prevent false positive decisions. Selective and sensitive fluorescence “off-on” sensors based on QDs have been widely applied for detection of melamine [15], cyanide [16], biothiol [17], nuclease [18], glutathione reductase activity [19], amino acid [20], virus [21] and DNA [22]. Shen et al. developed a fluorescent “offon” sensor based on (GSH)-capped CdTe for detection of hsDNA with Nile blue which is an intercalator molecule [23]. Vaishnavi and Renganathan have studied the interaction between positively charged
Corresponding author. E-mail address: [email protected] (O. Güney).
https://doi.org/10.1016/j.ab.2019.113540 Received 28 August 2019; Received in revised form 19 November 2019; Accepted 9 December 2019 Available online 13 December 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
Analytical Biochemistry 591 (2020) 113540
T. Arslan and O. Güney
porphyrin and ctDNA by using TGA-capped CdTe which is a fluorescent “off-on” probe [24]. Zhao et al. were investigated fluorescence “turnoff-on” method based on ZnCdSe QDs and they analyzed the interaction between TMPyP with dsDNA and ssDNA [25]. Molecularly imprinted polymer (MIP) is a multipurpose tool for the formation of specific binding sites with conforming the shape and size of the target molecules, correlating the code of interaction between lock and key [26,27]. MIPs are synthesized via polymerization procedure in the presence of template (target) molecules, functional monomers, cross-linkers, and then target molecule is removed from the polymer after synthesis [28,29]. MIP-coated QDs congregate the benefits of molecularly imprinted polymers and quantum dots, and exhibit significant change in fluorescent property upon interaction with target molecules [30,31]. MIP-QDs based ratiometric fluorescent sensors have notable attention because of their dependable selectivity and sensitivity in a trace amount of analyte [32,33]. Quenching of QDs fluorescence based on fluorescence resonance energy transfer (FRET) has some limitations compared to MIP-coated QDs. For instance, fluorescence of QDs is more affected by environmental conditions, inhomogeneous distributions and other organic quencher moieties in the sample, and also more susceptible to dynamic quenching [34,35]. Thus, MIP-coated QDs in a ratiometric analysis of dsDNA in a real sample has more advantages for accurate detection because of their relative change in uncalibrated signal ratios. The key advance of our current work involves the use of MIP-coated QDs for ratiometric detection of dsDNA and to our knowledge, based on turn off–on switch system by using MIP-coated QDs nanoparticles has not been reported before. In this work, we report a novel ratiometric sensing of dsDNA based on both RTP turn off–on switch system of molecularly imprinted polymer anchored on the surface of mercaptopropyltrimethoxysilane (MPTMS)-capped Mn-doped ZnS QDs (MIP-QDs) and fluorescence turnon of MG molecule. The complex is formed by MIP-QDs and MG via hydrogen bonding interaction, which leads to quenching of the RTP of MIP-QDs through phosphorescence resonance energy transfer (PRET), thereby ‘‘turning off’’ the RTP. By means of interaction with dsDNA, MG is forced to peel off from the surface of MIP-QDs and intercalates into the double helix structure of dsDNA so that the RTP of MIP-QDs can be restored and ‘‘turned on’‘. The RTP restoring degree of MIP-QDs and enhancement of MG fluorescence are gradually improved depending on increase in dsDNA content, so it can be designed as a new ratiometric sensor for quantitative and user-friendly DNA detection assay.
using a Varian Cary-Eclipse spectrophotometer controlled by a PC. VWR UV-1600PC Spectrometer was used for the recording of UV–visible spectra of samples. Fourier transform infrared (FTIR) of the samples were recorded on an Agilent Cary 630 FTIR spectrometer and pH of the solutions was measured with a VWR 730P pH meter. The sizes and shapes of MIP-QDs nanoparticles were investigated with an FEI Quanta 250 FEG model Scanning Electron Microscope (SEM) combined with Energy Dispersive X-ray (EDS) detector. X-ray diffraction (XRD) patterns were recorded by using A Bruker AXS D8 diffractometer (Cu Kα Cu Kα source (λ = 1.54184 Å) operated at 30 kV/10 mA) and the patterns were composed in the 5–15° 2θ range at a scan rate of 0.01° min−1. Transmission Electron Microscopy (TEM) images used for characterizing the surface morphology and size of samples as-prepared QDs and MIP-QDs were acquired on a JOEL 1220 JEM microscope. The TEM samples were prepared by dropping 5 μL of sample solution onto a carbon-coated Cu grid and drying at room temperature. Size distribution of the particles was determined using Dynamic Light Scattering technique (DLS) in Malvern Zeta-sizer Nano-ZS90 (Malvern Instruments Ltd, UK) using a standard rectangular quartz cell. 2.3. Synthesis of MPTMS-capped Mn-doped ZnS quantum dots Synthesis of Mn-doped ZnS quantum dots capped with MPTMS (QDs) was carried out based on our previous study with a minor modification [36]. Briefly, 1 mmol of MnCl2 and 12.5 mmol of ZnSO4 were dissolved in 40 mL water. Air in the system was removed by purging with nitrogen gas for 30 min at room temperature, and then 12.5 mmol of Na2S (10 mL) added dropwise into the former solution. After solution was stirred vigorously for the 30 min, 6.25 mmol of MPTMS (prepared in 10 mL of ethanol) was added drop by drop into the solution and reaction was continued for 24 h. QDs nanoparticles were precipitated with centrifugation and washed three times with both water and ethanol to eliminate the impurities. Finally, QDs nanoparticles were dried in a vacuum oven at 40 °C for 48 h. 2.4. Synthesis of MIP-QDs The MIP-QDs were obtained by sol-gel polymerization. Briefly, 33 mM of MG was dissolved in ethanol first and 0.1 M of APTES was thereafter added, and then the mixture was vigorously stirred for 30 min by permitting the self-unification of the MG and APTES. 100 mg of QDs and 0.4 M of TEOS were subsequently added and finally, 0.1 mL of NH3 solution was added to mixture. The all compounds were allowed to react for 16 h. At the end of the polymerization, MIP-QDs were washed to remove impurities and MG with methanol/acetic acid solution (9:1, v/v). Finally, synthesized MIP-QDs nanoparticles were dried under vacuum at 50 °C for 24 h (Fig. 1).
2. Experimental 2.1. Reagents and chemicals Tetraethyl orthosilicate (TEOS), (3-mercaptopropyl)trimethoxysilane (MPTMS) and (3-aminopropyl)triethoxysilane (APTES) were obtained from Alfa Aesar (Lancaster, UK). Malachite green, ammonium hydroxide, magnesium sulfate, calcium acetate, sodium nitrate, crystal violet, fuchsine and diamond fuchsine were purchased from Merck (Darmstadt, Germany). Sodium sulfide, theophylline, tryptophan, dopamine were purchased from Honeywell Fluka (Vantaa, Finland). Manganese (II) chloride tetrahydrate, zinc sulfate heptahydrate, ethidium bromide and double stranded DNA (dsDNA) sodium salt from salmon testes, which has ~2000 base pairs corresponding to an average molecular weight of 1.3 × 106 g/mol and melting temperature is 87.5 °C, were obtained from Sigma-Aldrich (Darmstadt, Germany). After the dsDNA was completely dissolved in phosphate buffer (10 mM) at pH 7.4, its concentration was determined using molar absorption coefficient (ε = 6600 M−1 cm−1) at 260 nm and the dsDNA stock solution was stored at 4 °C until further use.
3. Results and discussion 3.1. Structural characterization of QDs and MIP-QDs The presence of sol-gel imprinted polymer covering the exterior of QDs nanoparticles was confirmed by comparing FT-IR spectra of QDs and MIP-QDs (Fig. 2). As seen from Fig. 2, the existence of strong band around 1078 cm−1 for QDs and 1061 cm−1 for MIP-QDs remarked the asymmetric stretching of Si–O bond. The bands belonging to Si–O–Si bond were detected at 792 and 447 cm−1 (symmetric stretching and bending vibrations of Si–O, respectively). The peak around 1541 cm−1 (N–H band) denotes the existence of the amino functional group of silane molecule located on MIP-QDs, indicating the formation of the solgel network on the surface of QDs via condensation of silane molecules. The constituents of both QDs and MIP-QDs nanoparticles were estimated by obtaining the X-ray diffraction (XRD) of powder samples. As demonstrated in Fig. 3A, XRD patterns of the QDs nanoparticles defined as (111), (220) and (331) express the cubic phase of ZnS. XRD
2.2. Apparatus The phosphorescence and fluorescence spectra were obtained by 2
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T. Arslan and O. Güney
Fig. 1. Synthesis of QDs and MIP-QDs, and interaction with dsDNA.
measurements showed that the intensity of diffraction peak belonged to ZnS(111) covered by MG-imprinted polymer was lower than that of QDs, indicating the presence of sol-gel as amorphous material on the surface of MIP-QDs nanoparticles. In addition, EDS measurements also confirmed the XRD results. Furthermore, the detection of Zn, Mn and S elements which are essential constituents of Mn:ZnS QDs, the existence of C, O, Si and especially the N element peaks assure the coverage of sol-gel polymer on surface of QDs (Fig. 3B). Scanning electron microscopy (SEM) was used to evaluate the dimension and morphology of both QDs and MIPQDs nanoparticles (Fig. 4). SEM image of QDs revealed spherical morphology of QDs with a uniform in size (Fig. 4A). Fig. 4B displayed that MIP-QDs nanoparticles, with diameter distributions of 15–50 nm, bear some resemblance to QDs. The surface morphology and particle size distribution of the QDs and MIP-QDs samples are depicted in Fig. 5. The TEM image of QDs reveals that nanoparticles are mono-dispersed and uniform size without prominent aggregation (Fig. 5A). In contrast, MIP-QDs constitute aggregation with diverse interface between nanoparticles since the aggregation of MIP-QDs originates from their concentrations in aqueous solution (Fig. 5B). The results of DLS measurements revealed that the mean sizes of QDs and MIP-QDs are 37 and 49 nm, respectively (Fig. 5C and D). The
Fig. 2. FTIR spectra of QDs (a) and MIP-QDs (b).
Fig. 3. (A) XRD patterns of (a) QDs and (b) MIP-QDs. (B) EDS results of MIP-QDs nanoparticles. 3
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T. Arslan and O. Güney
Fig. 4. SEM images of nanoparticles; (A) QDs and (B) MIP-QDs.
solution in the absence and the presence of MG molecules, diminished as pH was reduced from 6.0 to 2.0 (Fig. S3). This is due to the instability of QDs nanoparticles in acidic pH values because of the production of H2S molecules [38]. RTP intensity of MIP-QDs at pH values higher than 8.0 likewise decreased since nucleophilic attack of hydroxyl groups creates defect on surface of QDs nanoparticles [39]. RTP intensity of MIP-QDs meanwhile was quenched in the presence of MG on a wide scale of pH values. However, enhancement in RTP intensity of MIP-QDs took place above pH 10 due to the weakening of the absorbance of MG molecules (Fig. S4).
size of MIP-QDs was higher than that of QDs, indicating that MIPs were synthesized successfully on QDs nanoparticles. 3.2. Spectral and optical characterization of MIP-QDs By varying the excitation wavelength, RTP spectra of MIP-QDs were recorded and maximum intensity was obtained at excitation wavelength of 320 nm (Fig. S1). MIP-QDs nanoparticles displayed phosphorescence peak at 594 nm due to the 4T 1 → 6 A1 transition because of Mn2+ impurity in QDs (Fig. S2). When the excitation wavelength increased, the slight shift in RTP peak maximum revealed the homogeneity in size of MIP-QDs nanoparticles [37]. RTP intensity of MIP-QDs, which was investigated upon pH of
Fig. 5. TEM images of the samples as-prepared QDs (A) and MIP-QDs (B) and particle size distribution of the QDs (C) and MIP-QDs (D) obtained from the DLS measurements. 4
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Fig. 6. (A) RTP spectra of MIP-QDs nanoparticles and (B) fluorescence spectra of MG containing dsDNA (10 μg/mL) for various MG concentration; (a) 0 μM, (b) 0.96 μM, (c) 1.9 μM, (d) 3.7 μM, (e) 7.4 μM, (f) 10.9 μM, (g) 17.5 μM, (i) 25.2 μM, (j) 32.2 μM, (k) 38.8 μM. (B). Insets: Change in emission intensities in RTP at 594 nm and fluorescence at 660 nm depending on concentration of MG.
3.3. Responses of MIP-QDs/dsDNA system to MG and fluorescence enhancement of MG
forced out of the molecular plane [43]. Hence, rotational motion and twisting of phenyl ring of MG lead to fast relaxation in a low viscous medium. Therefore, MG has low fluorescence quantum yield in a low viscous medium such as water, on the contrary, the quantum yield tends to increase in a high viscous medium because of restrict relaxation process of MG [44]. The fluorescence intensity at 660 nm exhibited increment with concentration of MG up to 7.0 μM, and then decreased depending on further increase in concentration (Fig. 6B, inset). This shows that maximum concentration of MG bound to dsDNA was 7.0 μM because higher concentrations of MG cause to inner filter effect due to the spectral overlap between the fluorescence and the absorption spectra of MG molecule. On the other hand, maximum concentration of MG bound to dsDNA was found to be 10 μM in the absence of MIP-QDs (Fig. S8). Therefore, MG concentration was chosen as 7.0 μM for further dsDNA titrations in the presence of MIP-QDs.
The RTP spectra of MIP-QDs and fluorescence emission spectra of MG in the presence of dsDNA were recorded simultaneously depending on increase in MG concentration (Fig. 6). The RTP intensity of MIP-QDs was gradually decreased due to the energy transfer between MIP-QDs and MG. At the same time, blue-shift in peak maximum from 594 nm to 581 nm was observed upon increase in MG concentration (Fig. 6A). Meanwhile, MIP-QDs displayed higher quenching efficiency toward MG when the titration was performed in the absence of dsDNA (Fig. S5). On the other hand, quenching data obtained in the absence and the presence of dsDNA were fitted to the Stern-Volmer equation (1) to elucidate the quenching constant of MG (Fig. S6).
P0/ P = 1 + K SV [Q]
(1)
where P0 is the RTP intensity of MIP-QDs; P is the RTP intensity after addition of MG; [Q] is the concentration of MG and KSV is the quenching constant. Stern-Volmer plot of the MIP-QDs exhibited linear relationship in the concentration range of between 0.1 and 40 μM. The value of KSV was found to be 5.82x105 and 3.88x105 L mol−1 in the absence and the presence of dsDNA, respectively. The results indicate that MG has residuary quenching effect on RTP of MIP-QDs in the presence of dsDNA due to the molecularly imprinted cavities existed on the surface for specific binding of MG, meaning that the mechanism behind the quenching is static rather than dynamic quenching. MG with cationic character may quench the RTP emission of the MIP-QDs through a charge transfer process since the band gap of MIPQDs is close to the absorption band edge of MG [40]. The electrons may transfer from the conduction and defect bands of the MIP-QDs to the lowest unoccupied molecular orbital (LUMO) of the visible band of the MG molecule since the absorption wavelength of MG at 615 nm is close to the emission wavelength of 4 T1 to 6 A1 transition of Mn2+ at 594 nm (Fig. S2). Moreover, due to the overall overlap of RTP spectrum of MIPQDs with the absorption spectrum of MG (Fig. S7), the phosphorescence resonance energy transfer (PRET) also contributed to the quenching of RTP emission of MIP-QDs by MG [41,42]. On the other hand, fluorescence emission spectra belonging to MG appeared upon increase in MG concentration in the presence of MIP-QDs and dsDNA (Fig. 6B). MG in fact is a non-fluorescent chromophoric dye, however the sharp fluorescence signal is observed at 650 nm when it interacts with dsDNA through intercalation. Furthermore, the fluorescence spectrum of MG exhibited bathochromic shift from 650 nm to 668 nm as a function of MG concentration (Fig. 6B). The distinct increase in fluorescence intensity and bathochromic shift are due to the increase in rigidity of the MG structure upon binding to the dsDNA. Because of the phenyl rings of MG twisting like propeller around to the central carbon atom, they are
3.4. DNA concentration-dependent fluorescence of MG and RTP of MIPQDs Both fluorescence and RTP spectra were obtained simultaneously upon addition of different amounts of dsDNA into the solution of MIPQDs/MG nanohybrids (Fig. 7). As shown in Fig. 7A, the fluorescence intensity of MG at 660 nm increased gradually upon the addition of dsDNA. Spectral shift was not observed in the fluorescence spectrum of MG with increase in dsDNA concentration since there was no concentrationdependent rigidity difference due to the presence of excess MG molecules. The enhancement of fluorescence intensity of MG depending on the concentration of dsDNA indicated that dsDNA could restrict the intra-molecular rotation of MG molecule (Fig. 7A, inset) [45]. Upon addition of dsDNA, the restoration of RTP of MIP-QDs in the presence of MG was observed (Fig. 7B), indicating that MG molecule bound to MIP-QDs peeled off in the presence of dsDNA. There was no spectral shift even at high concentrations of dsDNA. As seen from inset in Fig. 7B, the enhancement behavior in RTP is proportional to the concentration of dsDNA ranging from 0.1 to 4.0 μg/mL and can be monitored for the determination of dsDNA concentration. After titrations were completed, the photos of sample solutions of MG, MG/dsDNA, MIP-QDs and MIP-QDs/MG/dsDNA were taken under the ambient light and 400 nm laser beams. As seen from Fig. 8, the blue color of MG under ambient light turns into greenish blue upon addition of dsDNA and displays green fluorescence under the 400 nm laser beams. On the other hand, colorless and transparent solution of MIPQDs under ambient light exhibits phosphorescence under the 400 nm laser beams (Fig. 8, photo C, in the right). As seen from photo D in the right, slightly opacity and green color indicate the both of RTP and 5
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Fig. 7. (A) Fluorescence spectra of MG (7.0 μM) in the presence of MIP-QDs (0.1 mg/mL) upon addition of dsDNA in buffer (pH 7.4). Inset: Change in fluorescence emission intensity of MG at 660 nm depending on dsDNA concentration. (B) RTP spectra of MIP-QDs in the presence of MG as a function of dsDNA concentrations; a) 0, b) 0.6, c) 1.2, d) 1.8, e) 2.4, f) 3.6, g) 4.8, h) 7.2, i) 9.6 μg/mL. Inset: Change in RTP intensity of MIP-QDs at 594 nm with dsDNA addition.
Fig. 8. The photos of sample solutions; (A) MG, (B) MG/dsDNA, (C) MIP-QDs and (D) MIP-QDs/MG/dsDNA systems under the ambient light (in the left) and 400 nm laser beams (in the right).
Fig. 9. Change in RTP spectra of MIP-QDs (0.1 mg/mL)/dsDNA (10 μg/mL) hybrid nanoparticles in the presence of MG (4.0 μM) (A) and fluorescence spectra of MG (B) upon varying temperature. Insets: Change in RTP and fluorescence intensity depending on temperature.
dominate the involved mechanism. Furthermore, the enhanced fluorescence of MG in the presence of MIP-QDs/dsDNA nanohybrids decreases linearly with increase in temperature, indicating that binding of MG to dsDNA is intercalative rather than electrostatic (Fig. 9B).
fluorescence emissions from MIP-QDs/MG/dsDNA system. Temperature-dependent change in RTP and fluorescence of MIPQDs/MG/dsDNA nanohybrids system was simultaneously monitored to elucidate the mechanism behind quenching process (Fig. 9). As seen from Fig. 9A, the quenching of RTP of MIP-QDs slightly increased up to the temperature of 80 °C, where double helix of DNA unwinds to form two single strands. This is likely due to fact that intercalated MG molecules peel off from DNA and lead to pronounced quenching above 80 °C. This result shows that static rather than dynamic quenching processes might
3.5. Effect of interferences on MIP-QDs/MG/dsDNA system To evaluate the selectivity of the sensory system based on RTP recovery of MIP-QDs and fluorescence enhancement of MG molecule, we performed identical experiments by using both QDs/MG/dsDNA and 6
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Table 2 Recovery of dsDNA by nanohybrids sensor in urine sample. Urine sample
Added DNA (μg/ mL)
Found DNAa (μg/ mL)
Recovery (%)
RSD (%)
1 2 3
0.5 1.0 1.5
0.57 ± 0.018 1.05 ± 0.029 1.54 ± 0.032
106.0 105.0 102.6
3.15 2.76 2.07
a
Average three replicate determinations (mean ± SD).
biomolecules like theophylline (Thoe), tryptophan (Trp), dopamine (Dopa) were investigated. From the bar graph shown in Fig. 10, it seems that the influence of inorganic salts on both QDs and MIP-QDs system in the presence of dsDNA and MG is less even at relatively higher concentrations (10 mM). On the other hand, cationic dyes (concentration of 1.0 mM) and biomolecules (concentration of 10 mM) show prominent influence on the ratio of RTP594/F660 of QDs compared with MIP-QDs sensor system, indicating that biomolecules and cationic dyes were incapable to diffuse into the pores of MIP-QDs due to the unsuitable of molecular structure for recognition cavities. Therefore, the sensor system containing molecularly imprinted QDs (MIP-QDs) provides a more selective and novel ratiometric determination of dsDNA in physiological pH.
Fig. 10. The response of QDs (0.1 mg/mL) and MIP-QDs (0.1 mg/mL) in the presence of MG (7.0 μM) and dsDNA (2.0 μg/mL) and other interfering species at pH 7.4.
3.6. Ratiometric analysis of dsDNA with MIP-QDs/MG nanohybrids system To elucidate the linear range and LOD for the determination of dsDNA, we have conducted the titration experiment using diluted dsDNA stock solution (Fig. S10). Data obtained from fluorescence and RTP intensities as a function of dsDNA concentration were used to figure out the calibration curve (Fig. 11). The detection limit was evaluated based on formula: LOD = 3σ/K, where σ is the standard deviation and K is slope between F660/RTP594 ratio versus dsDNA concentration. The limit of detection (19.4 ng/mL) and linear range (0.089–1.79 μg/mL) were calculated from the y = 0.6244x + 0.2272 (R2 = 0.9914) linear equation. Although the higher limit of detection and narrower linear range as compared to other methods shown in Table 1, developed MIP-QDs/MG sensor system is weakly affected from back ground interference and can sense content of dsDNA selectively in a complex mixture without pretreatment. 3.7. Real sample measurements Fig. 11. Calibration curve for determination of dsDNA based on intensity ratio of MG fluorescence emission at 660 nm to RTP emission of MIP-QDs at 594 nm.
To validate the applicability of the proposed sensor system, urine was collected from healthy volunteer and analyzed for dsDNA. The urine sample solution was prepared by successively addition of MG solution (70 μL, 1.0 mM), MIP-QDs (0.1 mg/mL) and 0.1 mL of urine into a tube and then diluted with 10 mM of PBS solution to 10 mL. The spiked amounts of dsDNA in each different urine solution were 0.5, 1.0 and 1.5 μg/ml separately. Both RTP of MIP-QDs and fluorescence emission of MG after spiking of dsDNA were measured using the
MIP-QDs/MG/dsDNA systems. The impacts of some interfering inorganic salts such as magnesium sulfate (MgSO4), calcium acetate (Ca (Ac)2), sodium nitrate (NaNO3) and some cationic dyes which are analogues to MG molecule like crystal violet (Cv), fuchsine (Fu), ethidium bromide (EtBr), diamond fuchsine (Dfu) (Fig. S9), and also
Table 1 Comparison of ratiometric sensor performances for dsDNA detection with different optical schemes. Method
Systems for Sensing DNA
Linear Range (μg mL−1)
LOD (ng mL−1)
References
FL FL FL Cyclic Voltammetry FL RRS RTP RTP RTP FL/RTP
(GSH)-capped CdTe and Sm3+ (GSH) capped CdTe and (Pr3+-rutin) complex. TGA–CdTe/CdS QDs and daunorubicin AuNTsA electrode and methylene blue Glyp-functionalized-CdTe/CdS QDS
0.012–14.0 0.087–20.0 1.380–28.0 0.01–100 0.109–70 0.482–90 0.033–20 0.08–12 0.2–20 0.089–1.79
3.61 26.2 410 50 32.7 146 33 33.6 113 19.4
[46] [47] [48] [49] [22]
(MPA)-capped Mn:ZnS and Acridine Orange Mn-doped ZnS QDs and Methyl violet (MPA)-capped Mn:ZnS and Methylene Blue MIP-QDs and Malachite Green
7
[10] [50] [51] This work
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method in this system. As seen from Table 2, the recoveries of dsDNA for each of the urine sample were found to be in the range between 102.6 and 106% with RSDs of 2.66, indicating that nanohybrids sensor system could effectively detect dsDNA in urine without pretreatment for all samples.
[13]
[14]
4. Conclusions
[15]
In summary, a novel and selective method for the detection of dsDNA had been established. We have developed a system for ratiometric sensing of dsDNA based on both restoration of RTP of MIP-QDs complexed with energy-accepting MG molecules, and also fluorescence enhancement of MG embed into dsDNA duplexes generating more stable complex through intercalation binding. It was found that specific recognition of MG by MIP-QDs is essential towards selective determination of dsDNA. The use of the MIP-QDs as turn-on RTP sensor for quantitative detection of dsDNA was also demonstrated. Studies revealed that interferences caused by cationic dyes and organic matters hardly had effect on sensor system based on only QDs for the detection of dsDNA but encapsulation of QDs with molecularly imprinted polymer matrix supplied a novel platform for selective determination of dsDNA. The ratio of fluorescence enhancement of MG molecule to recovery of RTP of MIP-QDs provided a high selectivity for dsDNA detection with a good correlation coefficient and low detection limit.
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
CRediT authorship contribution statement [24]
Taner Arslan: Methodology, Validation, Investigation, Formal analysis. Orhan Güney: Supervision, Visualization, Writing - original draft, Writing - review & editing.
[25]
Appendix A. Supplementary data
[26]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ab.2019.113540.
[27]
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Ratiometric sensor based on imprinted quantum dots-cationic dye nanohybrids for selective sensing of dsDNA
T
Taner Arslan, Orhan Güney∗ Istanbul Technical University, Departments of Chemistry, 34469, Maslak, Istanbul, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Quantum dots Ratiometric sensor Surface imprinting dsDNA detection
A ratiometrically responsive sensor for dsDNA is reported, based on molecularly imprinted polymer coated quantum dots (MIP-QDs). A new platform is described for probing dsDNA by tracing the “turn on” fluorescence signal of malachite green (MG) as a cationic dye and “turn off-on” room temperature phosphorescence (RTP) signal of MIP-QDs/MG nanohybrids. The interaction between MIP-QDs surface and MG discloses an intense quenching in RTP (turning off) by a phosphorescence resonance energy transfer (PRET) process. After the addition of dsDNA, MG molecules escape from the MIP-QDs surface and intercalate into the dsDNA, resulting in the restoration of RTP intensity of MIP-QDs (turning on) and also enhancing in fluorescence of MG. This outcome hereby can be employed for the selective sensing of dsDNA via optical response. The ratio of fluorescence enhancement of MG to RTP intensity of MIP-QDs is proportional to the concentration of dsDNA in the range of 0.089–1.79 μg/mL with a detection limit (3σ/K) of 19.48 ng/mL under the optimized experimental conditions.
1. Introduction As the basic hereditary material in humans and almost all other organisms, DNA is crucial for the transportation of genetic information and the determinant of species continuation. Even slight change in structure of double-stranded DNA (dsDNA) may initiate the alteration of genetic characteristics and the occurrence of various diseases [1]. The level of DNA concentration in human plasma/serum might be sign of a variety of tumors and determination of DNA in urine could assist for early recognition of cancer, tuberculosis, HIV, malaria and potentially many other diseases [2]. Selective, sensitive and cost-effective detection of DNA has great significance in the diverse field including food safety, genetic diseases, pathology, pharmacokinetics, clinical diagnosis and treatments [3]. Quantitative detection of DNA has an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis. Currently, DNA diagnostic techniques are routinely used not only in research laboratories, but also in clinical and forensic practice. In the past decade, several strategies have been developed for DNA assay with including electrochemistry [4], surface-enhanced Raman spectroscopy (SERs) [5], colorimetric [6] and fluorescence methods [7]. Owing to the innate advantages, luminescence methods are fast, simple and high sensitive for DNA detection. Chromophoric dyes such as ethidium bromide, acridine orange and malachite green can form a
∗
stable fluorescent complex via intercalating with dsDNA [8–10]. Luminescence methods based on single emission could be faced by some interfering factors such as concentration of luminophore, output of instrument and specification of conditions. On the other hand, the ratiometric dual-emission can elude these effects and determine the analytes in complex samples through recording the ratios of emission intensities at two different wavelengths [11]. There are two main approaches for DNA analysis based on QDs; one way is “turn off” method (fluorescence quenching) and another way is “turn off-on” method (fluorescence quenching recovery) [10,12]. Analysis of DNA can be performed quickly by fluorescence “turn off” method, but false positive results may occur because non-specific disturbances caused by other quenchers in the medium instead of the analyte will result in a “turn off” state [13]. Furthermore, fluorescence “turn off-on” sensors have more selectivity than “turn off” methods [14]. Therefore, fluorescent “off-on” sensors provide more dependable detection performance and prevent false positive decisions. Selective and sensitive fluorescence “off-on” sensors based on QDs have been widely applied for detection of melamine [15], cyanide [16], biothiol [17], nuclease [18], glutathione reductase activity [19], amino acid [20], virus [21] and DNA [22]. Shen et al. developed a fluorescent “offon” sensor based on (GSH)-capped CdTe for detection of hsDNA with Nile blue which is an intercalator molecule [23]. Vaishnavi and Renganathan have studied the interaction between positively charged
Corresponding author. E-mail address: [email protected] (O. Güney).
https://doi.org/10.1016/j.ab.2019.113540 Received 28 August 2019; Received in revised form 19 November 2019; Accepted 9 December 2019 Available online 13 December 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
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porphyrin and ctDNA by using TGA-capped CdTe which is a fluorescent “off-on” probe [24]. Zhao et al. were investigated fluorescence “turnoff-on” method based on ZnCdSe QDs and they analyzed the interaction between TMPyP with dsDNA and ssDNA [25]. Molecularly imprinted polymer (MIP) is a multipurpose tool for the formation of specific binding sites with conforming the shape and size of the target molecules, correlating the code of interaction between lock and key [26,27]. MIPs are synthesized via polymerization procedure in the presence of template (target) molecules, functional monomers, cross-linkers, and then target molecule is removed from the polymer after synthesis [28,29]. MIP-coated QDs congregate the benefits of molecularly imprinted polymers and quantum dots, and exhibit significant change in fluorescent property upon interaction with target molecules [30,31]. MIP-QDs based ratiometric fluorescent sensors have notable attention because of their dependable selectivity and sensitivity in a trace amount of analyte [32,33]. Quenching of QDs fluorescence based on fluorescence resonance energy transfer (FRET) has some limitations compared to MIP-coated QDs. For instance, fluorescence of QDs is more affected by environmental conditions, inhomogeneous distributions and other organic quencher moieties in the sample, and also more susceptible to dynamic quenching [34,35]. Thus, MIP-coated QDs in a ratiometric analysis of dsDNA in a real sample has more advantages for accurate detection because of their relative change in uncalibrated signal ratios. The key advance of our current work involves the use of MIP-coated QDs for ratiometric detection of dsDNA and to our knowledge, based on turn off–on switch system by using MIP-coated QDs nanoparticles has not been reported before. In this work, we report a novel ratiometric sensing of dsDNA based on both RTP turn off–on switch system of molecularly imprinted polymer anchored on the surface of mercaptopropyltrimethoxysilane (MPTMS)-capped Mn-doped ZnS QDs (MIP-QDs) and fluorescence turnon of MG molecule. The complex is formed by MIP-QDs and MG via hydrogen bonding interaction, which leads to quenching of the RTP of MIP-QDs through phosphorescence resonance energy transfer (PRET), thereby ‘‘turning off’’ the RTP. By means of interaction with dsDNA, MG is forced to peel off from the surface of MIP-QDs and intercalates into the double helix structure of dsDNA so that the RTP of MIP-QDs can be restored and ‘‘turned on’‘. The RTP restoring degree of MIP-QDs and enhancement of MG fluorescence are gradually improved depending on increase in dsDNA content, so it can be designed as a new ratiometric sensor for quantitative and user-friendly DNA detection assay.
using a Varian Cary-Eclipse spectrophotometer controlled by a PC. VWR UV-1600PC Spectrometer was used for the recording of UV–visible spectra of samples. Fourier transform infrared (FTIR) of the samples were recorded on an Agilent Cary 630 FTIR spectrometer and pH of the solutions was measured with a VWR 730P pH meter. The sizes and shapes of MIP-QDs nanoparticles were investigated with an FEI Quanta 250 FEG model Scanning Electron Microscope (SEM) combined with Energy Dispersive X-ray (EDS) detector. X-ray diffraction (XRD) patterns were recorded by using A Bruker AXS D8 diffractometer (Cu Kα Cu Kα source (λ = 1.54184 Å) operated at 30 kV/10 mA) and the patterns were composed in the 5–15° 2θ range at a scan rate of 0.01° min−1. Transmission Electron Microscopy (TEM) images used for characterizing the surface morphology and size of samples as-prepared QDs and MIP-QDs were acquired on a JOEL 1220 JEM microscope. The TEM samples were prepared by dropping 5 μL of sample solution onto a carbon-coated Cu grid and drying at room temperature. Size distribution of the particles was determined using Dynamic Light Scattering technique (DLS) in Malvern Zeta-sizer Nano-ZS90 (Malvern Instruments Ltd, UK) using a standard rectangular quartz cell. 2.3. Synthesis of MPTMS-capped Mn-doped ZnS quantum dots Synthesis of Mn-doped ZnS quantum dots capped with MPTMS (QDs) was carried out based on our previous study with a minor modification [36]. Briefly, 1 mmol of MnCl2 and 12.5 mmol of ZnSO4 were dissolved in 40 mL water. Air in the system was removed by purging with nitrogen gas for 30 min at room temperature, and then 12.5 mmol of Na2S (10 mL) added dropwise into the former solution. After solution was stirred vigorously for the 30 min, 6.25 mmol of MPTMS (prepared in 10 mL of ethanol) was added drop by drop into the solution and reaction was continued for 24 h. QDs nanoparticles were precipitated with centrifugation and washed three times with both water and ethanol to eliminate the impurities. Finally, QDs nanoparticles were dried in a vacuum oven at 40 °C for 48 h. 2.4. Synthesis of MIP-QDs The MIP-QDs were obtained by sol-gel polymerization. Briefly, 33 mM of MG was dissolved in ethanol first and 0.1 M of APTES was thereafter added, and then the mixture was vigorously stirred for 30 min by permitting the self-unification of the MG and APTES. 100 mg of QDs and 0.4 M of TEOS were subsequently added and finally, 0.1 mL of NH3 solution was added to mixture. The all compounds were allowed to react for 16 h. At the end of the polymerization, MIP-QDs were washed to remove impurities and MG with methanol/acetic acid solution (9:1, v/v). Finally, synthesized MIP-QDs nanoparticles were dried under vacuum at 50 °C for 24 h (Fig. 1).
2. Experimental 2.1. Reagents and chemicals Tetraethyl orthosilicate (TEOS), (3-mercaptopropyl)trimethoxysilane (MPTMS) and (3-aminopropyl)triethoxysilane (APTES) were obtained from Alfa Aesar (Lancaster, UK). Malachite green, ammonium hydroxide, magnesium sulfate, calcium acetate, sodium nitrate, crystal violet, fuchsine and diamond fuchsine were purchased from Merck (Darmstadt, Germany). Sodium sulfide, theophylline, tryptophan, dopamine were purchased from Honeywell Fluka (Vantaa, Finland). Manganese (II) chloride tetrahydrate, zinc sulfate heptahydrate, ethidium bromide and double stranded DNA (dsDNA) sodium salt from salmon testes, which has ~2000 base pairs corresponding to an average molecular weight of 1.3 × 106 g/mol and melting temperature is 87.5 °C, were obtained from Sigma-Aldrich (Darmstadt, Germany). After the dsDNA was completely dissolved in phosphate buffer (10 mM) at pH 7.4, its concentration was determined using molar absorption coefficient (ε = 6600 M−1 cm−1) at 260 nm and the dsDNA stock solution was stored at 4 °C until further use.
3. Results and discussion 3.1. Structural characterization of QDs and MIP-QDs The presence of sol-gel imprinted polymer covering the exterior of QDs nanoparticles was confirmed by comparing FT-IR spectra of QDs and MIP-QDs (Fig. 2). As seen from Fig. 2, the existence of strong band around 1078 cm−1 for QDs and 1061 cm−1 for MIP-QDs remarked the asymmetric stretching of Si–O bond. The bands belonging to Si–O–Si bond were detected at 792 and 447 cm−1 (symmetric stretching and bending vibrations of Si–O, respectively). The peak around 1541 cm−1 (N–H band) denotes the existence of the amino functional group of silane molecule located on MIP-QDs, indicating the formation of the solgel network on the surface of QDs via condensation of silane molecules. The constituents of both QDs and MIP-QDs nanoparticles were estimated by obtaining the X-ray diffraction (XRD) of powder samples. As demonstrated in Fig. 3A, XRD patterns of the QDs nanoparticles defined as (111), (220) and (331) express the cubic phase of ZnS. XRD
2.2. Apparatus The phosphorescence and fluorescence spectra were obtained by 2
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Fig. 1. Synthesis of QDs and MIP-QDs, and interaction with dsDNA.
measurements showed that the intensity of diffraction peak belonged to ZnS(111) covered by MG-imprinted polymer was lower than that of QDs, indicating the presence of sol-gel as amorphous material on the surface of MIP-QDs nanoparticles. In addition, EDS measurements also confirmed the XRD results. Furthermore, the detection of Zn, Mn and S elements which are essential constituents of Mn:ZnS QDs, the existence of C, O, Si and especially the N element peaks assure the coverage of sol-gel polymer on surface of QDs (Fig. 3B). Scanning electron microscopy (SEM) was used to evaluate the dimension and morphology of both QDs and MIPQDs nanoparticles (Fig. 4). SEM image of QDs revealed spherical morphology of QDs with a uniform in size (Fig. 4A). Fig. 4B displayed that MIP-QDs nanoparticles, with diameter distributions of 15–50 nm, bear some resemblance to QDs. The surface morphology and particle size distribution of the QDs and MIP-QDs samples are depicted in Fig. 5. The TEM image of QDs reveals that nanoparticles are mono-dispersed and uniform size without prominent aggregation (Fig. 5A). In contrast, MIP-QDs constitute aggregation with diverse interface between nanoparticles since the aggregation of MIP-QDs originates from their concentrations in aqueous solution (Fig. 5B). The results of DLS measurements revealed that the mean sizes of QDs and MIP-QDs are 37 and 49 nm, respectively (Fig. 5C and D). The
Fig. 2. FTIR spectra of QDs (a) and MIP-QDs (b).
Fig. 3. (A) XRD patterns of (a) QDs and (b) MIP-QDs. (B) EDS results of MIP-QDs nanoparticles. 3
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Fig. 4. SEM images of nanoparticles; (A) QDs and (B) MIP-QDs.
solution in the absence and the presence of MG molecules, diminished as pH was reduced from 6.0 to 2.0 (Fig. S3). This is due to the instability of QDs nanoparticles in acidic pH values because of the production of H2S molecules [38]. RTP intensity of MIP-QDs at pH values higher than 8.0 likewise decreased since nucleophilic attack of hydroxyl groups creates defect on surface of QDs nanoparticles [39]. RTP intensity of MIP-QDs meanwhile was quenched in the presence of MG on a wide scale of pH values. However, enhancement in RTP intensity of MIP-QDs took place above pH 10 due to the weakening of the absorbance of MG molecules (Fig. S4).
size of MIP-QDs was higher than that of QDs, indicating that MIPs were synthesized successfully on QDs nanoparticles. 3.2. Spectral and optical characterization of MIP-QDs By varying the excitation wavelength, RTP spectra of MIP-QDs were recorded and maximum intensity was obtained at excitation wavelength of 320 nm (Fig. S1). MIP-QDs nanoparticles displayed phosphorescence peak at 594 nm due to the 4T 1 → 6 A1 transition because of Mn2+ impurity in QDs (Fig. S2). When the excitation wavelength increased, the slight shift in RTP peak maximum revealed the homogeneity in size of MIP-QDs nanoparticles [37]. RTP intensity of MIP-QDs, which was investigated upon pH of
Fig. 5. TEM images of the samples as-prepared QDs (A) and MIP-QDs (B) and particle size distribution of the QDs (C) and MIP-QDs (D) obtained from the DLS measurements. 4
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Fig. 6. (A) RTP spectra of MIP-QDs nanoparticles and (B) fluorescence spectra of MG containing dsDNA (10 μg/mL) for various MG concentration; (a) 0 μM, (b) 0.96 μM, (c) 1.9 μM, (d) 3.7 μM, (e) 7.4 μM, (f) 10.9 μM, (g) 17.5 μM, (i) 25.2 μM, (j) 32.2 μM, (k) 38.8 μM. (B). Insets: Change in emission intensities in RTP at 594 nm and fluorescence at 660 nm depending on concentration of MG.
3.3. Responses of MIP-QDs/dsDNA system to MG and fluorescence enhancement of MG
forced out of the molecular plane [43]. Hence, rotational motion and twisting of phenyl ring of MG lead to fast relaxation in a low viscous medium. Therefore, MG has low fluorescence quantum yield in a low viscous medium such as water, on the contrary, the quantum yield tends to increase in a high viscous medium because of restrict relaxation process of MG [44]. The fluorescence intensity at 660 nm exhibited increment with concentration of MG up to 7.0 μM, and then decreased depending on further increase in concentration (Fig. 6B, inset). This shows that maximum concentration of MG bound to dsDNA was 7.0 μM because higher concentrations of MG cause to inner filter effect due to the spectral overlap between the fluorescence and the absorption spectra of MG molecule. On the other hand, maximum concentration of MG bound to dsDNA was found to be 10 μM in the absence of MIP-QDs (Fig. S8). Therefore, MG concentration was chosen as 7.0 μM for further dsDNA titrations in the presence of MIP-QDs.
The RTP spectra of MIP-QDs and fluorescence emission spectra of MG in the presence of dsDNA were recorded simultaneously depending on increase in MG concentration (Fig. 6). The RTP intensity of MIP-QDs was gradually decreased due to the energy transfer between MIP-QDs and MG. At the same time, blue-shift in peak maximum from 594 nm to 581 nm was observed upon increase in MG concentration (Fig. 6A). Meanwhile, MIP-QDs displayed higher quenching efficiency toward MG when the titration was performed in the absence of dsDNA (Fig. S5). On the other hand, quenching data obtained in the absence and the presence of dsDNA were fitted to the Stern-Volmer equation (1) to elucidate the quenching constant of MG (Fig. S6).
P0/ P = 1 + K SV [Q]
(1)
where P0 is the RTP intensity of MIP-QDs; P is the RTP intensity after addition of MG; [Q] is the concentration of MG and KSV is the quenching constant. Stern-Volmer plot of the MIP-QDs exhibited linear relationship in the concentration range of between 0.1 and 40 μM. The value of KSV was found to be 5.82x105 and 3.88x105 L mol−1 in the absence and the presence of dsDNA, respectively. The results indicate that MG has residuary quenching effect on RTP of MIP-QDs in the presence of dsDNA due to the molecularly imprinted cavities existed on the surface for specific binding of MG, meaning that the mechanism behind the quenching is static rather than dynamic quenching. MG with cationic character may quench the RTP emission of the MIP-QDs through a charge transfer process since the band gap of MIPQDs is close to the absorption band edge of MG [40]. The electrons may transfer from the conduction and defect bands of the MIP-QDs to the lowest unoccupied molecular orbital (LUMO) of the visible band of the MG molecule since the absorption wavelength of MG at 615 nm is close to the emission wavelength of 4 T1 to 6 A1 transition of Mn2+ at 594 nm (Fig. S2). Moreover, due to the overall overlap of RTP spectrum of MIPQDs with the absorption spectrum of MG (Fig. S7), the phosphorescence resonance energy transfer (PRET) also contributed to the quenching of RTP emission of MIP-QDs by MG [41,42]. On the other hand, fluorescence emission spectra belonging to MG appeared upon increase in MG concentration in the presence of MIP-QDs and dsDNA (Fig. 6B). MG in fact is a non-fluorescent chromophoric dye, however the sharp fluorescence signal is observed at 650 nm when it interacts with dsDNA through intercalation. Furthermore, the fluorescence spectrum of MG exhibited bathochromic shift from 650 nm to 668 nm as a function of MG concentration (Fig. 6B). The distinct increase in fluorescence intensity and bathochromic shift are due to the increase in rigidity of the MG structure upon binding to the dsDNA. Because of the phenyl rings of MG twisting like propeller around to the central carbon atom, they are
3.4. DNA concentration-dependent fluorescence of MG and RTP of MIPQDs Both fluorescence and RTP spectra were obtained simultaneously upon addition of different amounts of dsDNA into the solution of MIPQDs/MG nanohybrids (Fig. 7). As shown in Fig. 7A, the fluorescence intensity of MG at 660 nm increased gradually upon the addition of dsDNA. Spectral shift was not observed in the fluorescence spectrum of MG with increase in dsDNA concentration since there was no concentrationdependent rigidity difference due to the presence of excess MG molecules. The enhancement of fluorescence intensity of MG depending on the concentration of dsDNA indicated that dsDNA could restrict the intra-molecular rotation of MG molecule (Fig. 7A, inset) [45]. Upon addition of dsDNA, the restoration of RTP of MIP-QDs in the presence of MG was observed (Fig. 7B), indicating that MG molecule bound to MIP-QDs peeled off in the presence of dsDNA. There was no spectral shift even at high concentrations of dsDNA. As seen from inset in Fig. 7B, the enhancement behavior in RTP is proportional to the concentration of dsDNA ranging from 0.1 to 4.0 μg/mL and can be monitored for the determination of dsDNA concentration. After titrations were completed, the photos of sample solutions of MG, MG/dsDNA, MIP-QDs and MIP-QDs/MG/dsDNA were taken under the ambient light and 400 nm laser beams. As seen from Fig. 8, the blue color of MG under ambient light turns into greenish blue upon addition of dsDNA and displays green fluorescence under the 400 nm laser beams. On the other hand, colorless and transparent solution of MIPQDs under ambient light exhibits phosphorescence under the 400 nm laser beams (Fig. 8, photo C, in the right). As seen from photo D in the right, slightly opacity and green color indicate the both of RTP and 5
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Fig. 7. (A) Fluorescence spectra of MG (7.0 μM) in the presence of MIP-QDs (0.1 mg/mL) upon addition of dsDNA in buffer (pH 7.4). Inset: Change in fluorescence emission intensity of MG at 660 nm depending on dsDNA concentration. (B) RTP spectra of MIP-QDs in the presence of MG as a function of dsDNA concentrations; a) 0, b) 0.6, c) 1.2, d) 1.8, e) 2.4, f) 3.6, g) 4.8, h) 7.2, i) 9.6 μg/mL. Inset: Change in RTP intensity of MIP-QDs at 594 nm with dsDNA addition.
Fig. 8. The photos of sample solutions; (A) MG, (B) MG/dsDNA, (C) MIP-QDs and (D) MIP-QDs/MG/dsDNA systems under the ambient light (in the left) and 400 nm laser beams (in the right).
Fig. 9. Change in RTP spectra of MIP-QDs (0.1 mg/mL)/dsDNA (10 μg/mL) hybrid nanoparticles in the presence of MG (4.0 μM) (A) and fluorescence spectra of MG (B) upon varying temperature. Insets: Change in RTP and fluorescence intensity depending on temperature.
dominate the involved mechanism. Furthermore, the enhanced fluorescence of MG in the presence of MIP-QDs/dsDNA nanohybrids decreases linearly with increase in temperature, indicating that binding of MG to dsDNA is intercalative rather than electrostatic (Fig. 9B).
fluorescence emissions from MIP-QDs/MG/dsDNA system. Temperature-dependent change in RTP and fluorescence of MIPQDs/MG/dsDNA nanohybrids system was simultaneously monitored to elucidate the mechanism behind quenching process (Fig. 9). As seen from Fig. 9A, the quenching of RTP of MIP-QDs slightly increased up to the temperature of 80 °C, where double helix of DNA unwinds to form two single strands. This is likely due to fact that intercalated MG molecules peel off from DNA and lead to pronounced quenching above 80 °C. This result shows that static rather than dynamic quenching processes might
3.5. Effect of interferences on MIP-QDs/MG/dsDNA system To evaluate the selectivity of the sensory system based on RTP recovery of MIP-QDs and fluorescence enhancement of MG molecule, we performed identical experiments by using both QDs/MG/dsDNA and 6
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Table 2 Recovery of dsDNA by nanohybrids sensor in urine sample. Urine sample
Added DNA (μg/ mL)
Found DNAa (μg/ mL)
Recovery (%)
RSD (%)
1 2 3
0.5 1.0 1.5
0.57 ± 0.018 1.05 ± 0.029 1.54 ± 0.032
106.0 105.0 102.6
3.15 2.76 2.07
a
Average three replicate determinations (mean ± SD).
biomolecules like theophylline (Thoe), tryptophan (Trp), dopamine (Dopa) were investigated. From the bar graph shown in Fig. 10, it seems that the influence of inorganic salts on both QDs and MIP-QDs system in the presence of dsDNA and MG is less even at relatively higher concentrations (10 mM). On the other hand, cationic dyes (concentration of 1.0 mM) and biomolecules (concentration of 10 mM) show prominent influence on the ratio of RTP594/F660 of QDs compared with MIP-QDs sensor system, indicating that biomolecules and cationic dyes were incapable to diffuse into the pores of MIP-QDs due to the unsuitable of molecular structure for recognition cavities. Therefore, the sensor system containing molecularly imprinted QDs (MIP-QDs) provides a more selective and novel ratiometric determination of dsDNA in physiological pH.
Fig. 10. The response of QDs (0.1 mg/mL) and MIP-QDs (0.1 mg/mL) in the presence of MG (7.0 μM) and dsDNA (2.0 μg/mL) and other interfering species at pH 7.4.
3.6. Ratiometric analysis of dsDNA with MIP-QDs/MG nanohybrids system To elucidate the linear range and LOD for the determination of dsDNA, we have conducted the titration experiment using diluted dsDNA stock solution (Fig. S10). Data obtained from fluorescence and RTP intensities as a function of dsDNA concentration were used to figure out the calibration curve (Fig. 11). The detection limit was evaluated based on formula: LOD = 3σ/K, where σ is the standard deviation and K is slope between F660/RTP594 ratio versus dsDNA concentration. The limit of detection (19.4 ng/mL) and linear range (0.089–1.79 μg/mL) were calculated from the y = 0.6244x + 0.2272 (R2 = 0.9914) linear equation. Although the higher limit of detection and narrower linear range as compared to other methods shown in Table 1, developed MIP-QDs/MG sensor system is weakly affected from back ground interference and can sense content of dsDNA selectively in a complex mixture without pretreatment. 3.7. Real sample measurements Fig. 11. Calibration curve for determination of dsDNA based on intensity ratio of MG fluorescence emission at 660 nm to RTP emission of MIP-QDs at 594 nm.
To validate the applicability of the proposed sensor system, urine was collected from healthy volunteer and analyzed for dsDNA. The urine sample solution was prepared by successively addition of MG solution (70 μL, 1.0 mM), MIP-QDs (0.1 mg/mL) and 0.1 mL of urine into a tube and then diluted with 10 mM of PBS solution to 10 mL. The spiked amounts of dsDNA in each different urine solution were 0.5, 1.0 and 1.5 μg/ml separately. Both RTP of MIP-QDs and fluorescence emission of MG after spiking of dsDNA were measured using the
MIP-QDs/MG/dsDNA systems. The impacts of some interfering inorganic salts such as magnesium sulfate (MgSO4), calcium acetate (Ca (Ac)2), sodium nitrate (NaNO3) and some cationic dyes which are analogues to MG molecule like crystal violet (Cv), fuchsine (Fu), ethidium bromide (EtBr), diamond fuchsine (Dfu) (Fig. S9), and also
Table 1 Comparison of ratiometric sensor performances for dsDNA detection with different optical schemes. Method
Systems for Sensing DNA
Linear Range (μg mL−1)
LOD (ng mL−1)
References
FL FL FL Cyclic Voltammetry FL RRS RTP RTP RTP FL/RTP
(GSH)-capped CdTe and Sm3+ (GSH) capped CdTe and (Pr3+-rutin) complex. TGA–CdTe/CdS QDs and daunorubicin AuNTsA electrode and methylene blue Glyp-functionalized-CdTe/CdS QDS
0.012–14.0 0.087–20.0 1.380–28.0 0.01–100 0.109–70 0.482–90 0.033–20 0.08–12 0.2–20 0.089–1.79
3.61 26.2 410 50 32.7 146 33 33.6 113 19.4
[46] [47] [48] [49] [22]
(MPA)-capped Mn:ZnS and Acridine Orange Mn-doped ZnS QDs and Methyl violet (MPA)-capped Mn:ZnS and Methylene Blue MIP-QDs and Malachite Green
7
[10] [50] [51] This work
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method in this system. As seen from Table 2, the recoveries of dsDNA for each of the urine sample were found to be in the range between 102.6 and 106% with RSDs of 2.66, indicating that nanohybrids sensor system could effectively detect dsDNA in urine without pretreatment for all samples.
[13]
[14]
4. Conclusions
[15]
In summary, a novel and selective method for the detection of dsDNA had been established. We have developed a system for ratiometric sensing of dsDNA based on both restoration of RTP of MIP-QDs complexed with energy-accepting MG molecules, and also fluorescence enhancement of MG embed into dsDNA duplexes generating more stable complex through intercalation binding. It was found that specific recognition of MG by MIP-QDs is essential towards selective determination of dsDNA. The use of the MIP-QDs as turn-on RTP sensor for quantitative detection of dsDNA was also demonstrated. Studies revealed that interferences caused by cationic dyes and organic matters hardly had effect on sensor system based on only QDs for the detection of dsDNA but encapsulation of QDs with molecularly imprinted polymer matrix supplied a novel platform for selective determination of dsDNA. The ratio of fluorescence enhancement of MG molecule to recovery of RTP of MIP-QDs provided a high selectivity for dsDNA detection with a good correlation coefficient and low detection limit.
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
CRediT authorship contribution statement [24]
Taner Arslan: Methodology, Validation, Investigation, Formal analysis. Orhan Güney: Supervision, Visualization, Writing - original draft, Writing - review & editing.
[25]
Appendix A. Supplementary data
[26]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ab.2019.113540.
[27]
References
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