Room-Temperature Phosphorescence Turn-on

J Fluoresc DOI 10.1007/s10895-015-1699-6

ORIGINAL ARTICLE

Room-Temperature Phosphorescence Turn-on Detection of DNA Based on Riboflavin-Modulated Manganese Doped Zinc Sulfide Quantum Dots Yan Gong 1 & Zhefeng Fan 1

Received: 19 June 2015 / Accepted: 20 October 2015 # Springer Science+Business Media New York 2015

Abstract A sensitive phosphorescent sensor based on riboflavin (RF)-modulated mercaptopropionic acid (MPA)-capped Mn-doped ZnS quantum dots (QDs) was developed and utilized as room-temperature phosphorescence (RTP) sensor for DNA detection. The RTP of the MPA-capped Mn-doped ZnS QDs was stored via photoinduced electron transfer by RF, and formed an electrochemically nonactive QDs/RF nanohybrids through electrostatic attraction. In the presence of DNA, RF could bind with DNA, which has a double helical structure, via electrostatic interaction and intercalation. RF can be removed from the surface of the QDs, thus releasing the RTP of the QDs. On the basis of this principle, an RTP sensor for DNA detection was developed. Under optimal conditions, the detection limit for DNA was 15 μg mL−1, the relative standard deviation was 1.9 %, and the method recovery ranged from 97 % to 103 %. The proposed method was applied to biological fluids, in which satisfactory results were obtained. Keywords Quantum dots . Room-temperature phosphorescence . DNA . Riboflavin . Photoinduced electron transfer

Electronic supplementary material The online version of this article (doi:10.1007/s10895-015-1699-6) contains supplementary material, which is available to authorized users. * Zhefeng Fan [email protected] 1

Department of Chemistry, Shanxi Normal University, Linfen 041004, People’s Republic of China

Introduction Sensors based on quantum dots (QDs) for chemical and biological detections have gained considerable attention in the past decade [1–4] because of the unique properties exhibited by a variety of nanomaterials in conjugation with natural or artificial molecular recognition units [5–7]. Considering the phosphorescence of QDs generated by the short-lived singlet state and the phosphorescence from the triple state, phosphorescence has longer average life expectancy than fluorescence, which allows an appropriate delay time; in addition, any fluorescence emission and scattering light can be easily avoided [8]. Selectivity is also enhanced because phosphorescence is a less usual phenomenon than fluorescence [9]. Stimulated QDs could generate electrons or energy transfer between their donor and receptor, which results in quenching of their photoluminescence [10–12]. The combination of QDs with complementary receptors could lead to a significant change in the luminescence emission intensity of the QDs [13]. Sensors based on the QD-fluorescence resonance energy transfer has been applied to the analysis of protein-small molecule or protein-protein combination [14, 15], immunoassay [16], DNA hybridization probe [17, 18], and enzyme activity measurement [19]. Later, sensors based on the principle of QD-photoinduced electron transfer (PIET) have emerged [13, 20]. PIET-based mechanism has been speculated to rely on the electrostatic attraction between the quencher and the surface groups of QDs as a result of luminescent quenching, and the emission restoration by a receptor could bind with the quencher and remove it from the surface of QDs [21]. PIET-based sensors have been applied to the detection of glucose [22], pH value [23], maltose [24], permeability of organic monolayers [25], and anions/cations [26, 27]. Such studies are

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mostly based on the fluorescence properties of QDs, but PIET-based sensors that apply the room-temperature phosphorescence (RTP) properties of QDs have been rarely reported. Detection of trace DNA, as the most important biological macromolecule, is essential in the fields of pharmacogenetics, pathology, genetics, and food safety [28]. The concentration of DNA in human plasma/serum is suggested to be an indicator of a variety of tumors [29]. Many DNA sensors have been reported [30, 31], including electrochemical [32, 33], fluorescent [34], Raman spectroscopy [35], and chemiluminescence [36] methods. However, electrode modification or labeling process with chromophores is expensive, complex, and time consuming, which may limit its applications to detect nucleic acids [37]. The widely used fluorescence probes to detect DNA often suffer from photobleaching, low-signal intensities, and random on/off light emission. Therefore, construction of a sensitive and convenient DNA sensor is highly desirable. Riboflavin (RF) is a key component of the redox cofactors flavin adenine dinucleotide and flavin mononucleotide; RF plays an important role in vivo as electron transporter [38]. As an electron accepter, RF could quench the RTP of mercaptopropionic acid (MPA)-capped Mn-doped ZnS QDs via PIET and form Mn-doped ZnS QDs/RF nanohybrids by electrostatic attraction. RF also shows high affinity of DNA duplex; it can interact with DNA in groove-binding model by electrostatic effect and form an electrochemically nonactive complex [39]. In Mn-doped ZnS QDs/RF nanohybrids co-existing with DNA, RF will inlay in DNA double helix and be competitively removed from the surface of Mn-doped ZnS QDs, thereby releasing the RTP of Mn-doped ZnS QDs (Scheme 1a). Degree of recovery of Mn-doped ZnS QDs depends on DNA concentration. The developed QD-based RTP sensor acts in a turn-on mode and offers high sensitivity to DNA, and the interferences from background fluorescence and scattered light can be easily avoided. This method can be used for rapid and sensitive detection of DNA in biological fluids without chemical modification and fixation.

Corporation, Kansas City, MO). Riboflavin (RF) and Salmon Sperm DNA were provided by Sigma (Product of USA). Instrumentation A Cary Eclipse phosphorescence spectrophotometer with excitation wavelength at 295 nm (Varian, American), equipped with a plotter unit and a quartz cell (1.0 cm × 1.0 cm) was used in this study. The slit widths of excitation and emission were 10 and 20 nm for phosphorescence mode with an excitation wavelength of 295 nm and emission wavelength of 590 nm. Absolute quantum yield was measured using FLsp 920 fluorescence lifetime and steady-state spectrometer (Edinburgh Instruments, England) UV-visible absorption spectra were acquired using a UV-4100 spectrophotometer (Shimadzu, Janpan). The QDs were characterized by a JEM-2100 (JEOL, Japan) transmission electron microscope (TEM), and a D8 Advance (Bruker, Germany) X-ray diffractometer (Cu Kα). The samples for TEM were obtained by drying sample droplets from water dispersion onto a 100-mesh Cu grid coated with a lacey carbon film, which was then allowed to dry prior to imaging. The resonance light scattering spectra (RLS) were recorded in the same spectrofluorometer by simultaneously scanning the excitation and emission monochromators (Δλ = 0) from 200 to 700 nm. Zeta potential experiments were carried out on a Malvern Instruments Zetasizer 2000, Nano series- ZS (Malvern Instruments Ltd., UK) using a standard rectangular quartz cell. Synthesis of Aqueous MPA-Capped Mn-Doped ZnS QDs

Experimental Section

Synthesis of Mn-doped ZnS QDs was conducted in an aqueous solution in accordance with a published method with minor modification [40, 41]. Briefly, 5 mL of 0.1 M ZnSO4, 2 mL of 0.01 M MnCl2, and 50 mL of 0.04 M MPA were added to a three-neck flask. The pH of the mixed solution was adjusted to 11 with 1 M NaOH and stirred under Ar at room temperature for 30 min. Subsequently, 5 mL of 0.1 M Na2S was immediately injected into the mixture, stirred for 20 min, and aged at 50 °C under open air for 2 h to form MPA-capped Mn-doped ZnS QDs. The QDs were purified by precipitation with ethanol, separation by centrifugation, washing with ethanol, and drying in a vacuum. The obtained MPA-capped Mn-doped ZnS QD powder was highly soluble in water.

Materials and Chemicals

Assay Condition and RTP Measurement

Mercaptopropionic acid (MPA) (J&K Scientific, Beijing, China), ZnSO4·7H2O, MnCl2, and Na2S·9H2O (Tianjing Kermel Chemical Reagent Co., China) were used for the preparation of Mn-doped ZnS QDs, all chemicals used were of analytical reagent grade. Ultrapure water (18.2 MΩ cm) was obtained from a Water Pro water purification system (Labconco

To determine the effect of RF on the RTP intensity of the MPA-capped Mn-doped ZnS QDs, RF was dissolved in water to obtain a 10 mg L−1 solution. A series of samples with different concentrations were prepared by adding various amounts of RF to phosphate-buffered saline (PBS, pH 7.4, 20 mM). MPA-capped Mn-doped ZnS QDs were dissolved

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Scheme 1 a Schematic illustration of the RF-modulated MPA-capped Mn-doped ZnS QDs/nanohybrids for DNA detection;(b MPA-capped Mn-doped ZnS QDs/RF nanohybrids formed via PIET

in water to obtain a solution of 4.0 mg mL−1. The QD solution (100 μL) was added to each of the above RF solutions. After 10 min, phosphorescence was measured at an excitation wavelength of 295 nm. For DNA determination, DNA was dissolved in water to obtain a 1 mg mL−1 solution. Assay solutions that contained MPA-capped Mn-doped ZnS QDs (100 μL), RF (100 μL), and various concentrations of DNA (0 mg L−1–40 mg L−1) were prepared in 10 mL of PBS (20 mM, pH 7.4). The reactions were allowed to proceed for 15 min before spectrophotometric analysis. Sample Pretreatment The urine and serum samples were collected from healthy volunteer. Sample was subjected to a 100-fold dilution before detection and no other pretreatments were used. Measurement Procedures PBS (0.2 M, 0.5 mL), RF (10 mg L−1, 200 μL), MPA-capped Mn-doped ZnS QDs (4 mg mL−1, 100 μL), and urine were sequentially added to a 10 mL calibrated test tube. A recovery study was conducted in the samples spiked with 2.0, 5.0, and 10.0 mg L−1 DNA. The mixture was diluted to volume with ultrapure water, mixed thoroughly, aged for 15 min, and used for phosphorescence measurements at an excitation wavelength of 295 nm. Experiments were repeated three times.

No further pretreatment procedures were employed in sample preparation.

Results and Discussion Characterization of the MPA-Capped Mn-Doped ZnS QDs The XRD spectra were scaned over the 2 theta (θ) range from 10 to 80°, as shown in Fig. 1a, the XRD pattern of MPA-capped Mn-doped ZnS QDs exhibited a cubic structure, the diffractive peaks of the QDs at 28°, 46° and 57° indicated well-crystallized QDs were obtained by this method. Size characterization was carried out measuring the diameter of the QDs by transmission electron microscopy (TEM), as shown in the inset of Fig. 1a. The image reveals Mn-doped ZnS QDs was nearly monodispersed and have an average diameter of 3 nm. The as-prepared MPA-capped Mn-doped ZnS QDs exhibited the maximum excitation peak at 295 nm and a narrow emission band centered at 590 nm (Fig. 1b). The energy transfer from the band gap of ZnS to Mn2+ dopant and the subsequent transition from the triplet state (4T1) to the ground state (6A1) of the Mn2+ incorporated into the ZnS host lattice would result in an orange phosphorescence emission (about 590 nm) [42]. The MPA-capped Mn-doped ZnS QDs exhibit 26 % quantum yield and stable in water for at least 6 months without notable precipitation in the dark under ambient conditions.

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Fig. 1 a XRD image of MPA-capped Mn-doped ZnS QDs; And TEM image of the QDs (the inset); b The excitation and RTP emission spectra of Mn-doped ZnS QDs (40 mg L−1). All solutions were prepared in 20 mM PBS buffer at pH 7.4

Formation of MPA-Capped Mn-Doped ZnS QDs with RF Nanohybrids The MPA that capped the Mn-doped ZnS QDs could enhance the water solubility of QDs and endows negative charge on the surface of QDs. The zeta potentials of MPA-capped Mn-doped ZnS QDs and positively charged RF were measured to be −24.8 and 17.5 mV, respectively. The different charges between them formed QDs/RF nanohybrids via electrostatic attraction (Scheme 1b). The enhancement of the ultraviolet (UV) spectrum of QDs with different concentrations of RF without emission peak displacement indicated the formation of QDs/RF nanohybrids only through electrostatic attraction (Fig. 2a). As a good electron acceptor, PIET occurs between RF and QDs. Figure S1 shows that the RF concentration was dependent on the RTP intensity of Mn-doped ZnS QDs. The RTP intensity of Mn-doped ZnS QDs was quenched at 590 nm with the increased RF concentration and was basically

Fig. 2 UV-vis absorption spectra of DNA, RF, the QDs/RF nanohybrids and DNA (b); RF and DNA (c). Concentration of DNA and RF are 40 mg L−1 and 1 mg L−1 respectively

stabilized when the RF concentration reached 0.2 mg L−1 (inset in Fig. S1).

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Factors That Affect the Stability of Mn-Doped ZnS QDs/RF Nanohybrids The pH value of the solution greatly affected the RTP intensity of the QD/RF nanohybrid system. The RTP intensity increased with the increase in pH from 5.5 to 9.0 but was relatively stable at pH 7.0–8.0. A pH value of 7.4, close to that of biological fluid, was selected for further experiments. The RTP intensity of the nanohybrids was basically stable within 10 min (Fig. S2). The concentration of NaCl in the range from 0 M to 0.1 M had no influence on the RTP emission of the nanohybrids. When the concentration of NaCl was higher than 0.1 M, the RTP emission of the nanohybrids increased since charges of the system could be screened and the interaction via electrostatic force will be suppressed. The RF-Modulated Mn-Doped ZnS QDs as an RTP Probe for DNA

DNA concentration (Fig. 3), which validated the feasibility of the QDs/RF nanohybrids as the RTP probe of DNA. Figure S3 shows the resonance light scattering (RLS) spectra of DNA-RF interaction. After DNA addition, the RLS intensity of RF was gradually enhanced with the increase in DNA concentration, which indicated the occurrence of interaction and combination of RF and DNA. Figure 4a shows the fluorescence spectrum of RF in the presence of QDs. When QDs were added into RF, no enhancement in the superimposed fluorescence spectrum occurred. However, the fluorescence intensity significantly decreased, the maximum emission peak position was essentially the same. Given that the hydroxyl group on RF interacted with the carboxyl on the surface of the QDs, the changed substituent of RF could weaken the coplanarity between n electron cloud and π electron cloud in the ring, thereby reducing the possible formation of conjugated π bond; n-π* transitions

The ultraviolet (UV) spectra of DNA, RF, MPA-capped Mn-doped ZnS QDs and the interaction among them as shown in Fig. 2. The UV spectra of Mn-doped ZnS QDs show enhancement (without any shift) with the elevated concentration of RF (Fig. 2a). After addition of DNA into the solution of Mn-doped ZnS QDs/RF nanohybrids, the UV spectra of the nanohybrids showed further increased with slight blue-shift (Fig. 2b). The UV spectra of RF with addition of DNA produce a significant subtractive effect and red-shift (Fig. 2c). These phenomena indicate that the interaction among the MPA-capped Mn-doped ZnS QDs, RF, the QDs/RF nanohybrids and DNA indeed occur. The RTP intensity of the Mn-doped ZnS QDs/RF nanohybrids was gradually enhanced with the increase of

Fig. 3 RTP spectra of MPA-capped Mn-doped ZnS QDs/RF (40 mg L−1) nanohybrids in the presence of DNA at various concentrations (0, 0.02, 0.1, 0.8, 1.5, 5.0, 12, 18, 20 mg L−1). All solutions were prepared in 20 mM PBS buffer at pH 7.4

Fig. 4 (a) Fluorescence spectrum of RF (1 mg L−1) with different concentration of MPA-capped Mn-doped ZnS QDs (0, 5.0, 10, 15 and 20 mg L−1). (b) The fluorescence spectrum of RF (0.1 mg L−1) with different concentration of DNA (0, 5, 15, 20 and 40 mg L−1)

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belong to forbidden transition. This process affected the luminescent properties of RF, which reduced the fluorescence intensity. The nanohybrids can be used as sensor for DNA detection owing to the high sensitivity of the RTP intensity of QDs to RF at trace level, the relative stability of the QDs/RF nanohybrids, and the good binding capability between RF and DNA. Working Mechanism Between DNA and RF RF had a strong fluorescence emission peak at 528 nm (Fig. 4b). With the addition of DNA, the fluorescence intensity of RF at 528 nm was quenched without emission peak displacement. This phenomenon indicated that RF could inlay in DNA double helix through H bonds because RF have a triple ring plane structure, −N-, and -O- on the ring readily formed H bonds. The intercalative binding could result in a charge transfer and reduce the chance of π-π* transition, and the change of electron energy in excited state finally led to fluorescence quenching. Figure 5a shows the Stern-Volmer’s curve representing the quenching effect of DNA on RF fluorescence intensity. The relative fluorescence intensity F0/F (F0 and F is the FL intensity of RF in the absence/presence of DNA respectively) exhibited a linear relationship with the concentration of DNA (CDNA) in the range from 15 μg L−1 to 40 mg L−1 with the linear regression equation of F0/F = 1.0 + 2.8 × 103 CDNA (R = 0.998). According to Stern-Volmer’s equation [43], F0/F = 1 + KSVCDNA = 1 + Kqτ0CDNA −8

where τ0 is fluorescence lifetime of RF [44] about 10 s, KSV is the quenching constant 2.8 × 103 L mol−1. Kq (2.8 × 1011 L mol−1 S−1) is the rate constant of bimolecular quenching process, can be calculated from Stern-Volmer’s equation. The value of Kq is greater than the collision coefficient of maximum diffusion control (2.0 × 1010 L mol−1 S−1) between small molecules and biological macromolecules. Therefore, the quenching effect of DNA on RF is not dynamic quenching, but static quenching by forming ground state complex. According to static quenching theory, the equation 1 g(F0/ F-1) = 1gKA + nlgCDNA is applicable, KA(L mol −1) is the binding constant between RF and DNA, and n is the binding site size. The binding constant (KA) with 8.9 × 103 L mol−1 could be obtained from the graph of 1 g(F0/F-1) plotted versus lgCDNA (Fig. 5b), and the binding site size was determined to be 1.3. The results indicated that RF could bind with DNA at a ratio of 1:1. The UV absorption spectrum of DNA changes after it interacts with other compounds. Hyperchromic and subtractive effects of DNA are the unique spectral properties of the DNA

Fig. 5 The standard curve of F0/F plotted versus CDNA (a) and 1 g(F0/F1) plotted versus lgCDNA (B). All solutions were prepared in 20 mM PBS buffer at pH 7.4

double-helix structure. The hyperchromic effect is the result of the destroyed DNA double-helix structure, whereas the subtractive effect is caused by the molecule axial contraction and conformational change in DNA [45]. As shown in Fig. 2c, the peaks at 223.0, 267.0, 372.0, and 445.0 nm in the UV spectra are attributed to RF. After DNA addition, slightly redshifted peaks were observed, accompanied by a significant subtractive effect. The obtained UV spectra of the interaction between RF and DNA in the experiment were much less than the superposition of the theoretical value. Therefore, RF embedded the DNA base pairs and readily interacted with the base pairs, which changed the UV spectra [45]. The UV spectra of RF gradually decreased with DNA addition. This phenomenon was attributed to the π electron stacking caused by the intercalating effect between RF and DNA double helix. The reduced energy caused by π* vacant orbit of RF coupling with π orbital of DNA bases decreased π-π* transition energy. A subtractive

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effect was then generated because the probability of π-π* transition was reduced owing to the coupled π* orbital partially filled with electrons. The results indicated that the interaction between DNA and RF relied on electrostatic attraction and molecular intercalation, whereas the interaction between Mn-doped ZnS QDs and RF only relied on electrostatic attraction. Thus, the interaction of RF with DNA was superior to that of RF with Mn-doped ZnS QDs. DNA could seize RF from the Mn-doped ZnS QDs/RF nanohybrids, thus leading to the recovery of RTP in Mn-doped ZnS QDs.

Characterization of the RF-Modulated Mn-Doped ZnS QDs as RTP Probes To assess the potential of Mn-doped ZnS QD/RF nanohybrid RTP sensor for DNA detection, the RTP intensity of Mn-doped ZnS QDs/RF nanohybrids was measured (emission wavelength at 590 nm) as a function of DNA concentration (Fig. 3). Under optimal condition, the RTP quenching value (ΔRTP) of Mn-doped ZnS QDs/RF nanohybrids exhibited a linear relationship with the concentration of DNA (CDNA) in the range from 0.02 mg L−1 to 20 mg L−1, with the linear equation of ΔRTP =29.51CDNA + 15.271 (R = 0.998). For systems without DNA and with 0.5 mg L−1 DNA, the 11 continuous parallel detections on phosphorescence intensity had a relative standard deviation (RSD) of 1.9 % and with a detection limit of 15 μg L−1.

Factors That Affect the RTP Sensing of DNA Based on the RF-Modulated Mn-Doped ZnS QDs The pH of the solution greatly affected the increased RTP intensity of the system. The recovered RTP intensity significantly changed when the pH was lower than 7.0 or higher than 7.5. The optimal pH range is 7.0 to 7.5. A pH value of 7.4, close to that of biological fluid, was selected for further experiments. To assess the stability of the RTP sensing system, we investigated the time-dependence of the RTP intensity of the Mn-doped ZnS QDs/RF nanohybrids at pH 7.4. Figure S2 shows that after addition of RF into Mn-doped ZnS QDs, the RTP intensity of Mn-doped ZnS QDs quenched immediately and remained constant for 10 min, then be recovered by DNA in 15 min. The concentration of NaCl in the range from 0 M to 0.1 M showed no influence on the RTP recovery of the nanohybrids. When the concentration of NaCl was higher than 0.1 M, the efficiency of recovered RTP was reduced because charges could be screened, and the interaction via electrostatic force is weakened.

Selectivity of the RF-Modulated Mn-Doped ZnS QDs RTP Probes Several common metal ions and biomolecules in biological fluids were used to investigate their interferences on DNA detection by Mn-doped ZnS QD/RF nanohybrid RTP probes. The restored RTP with 0.5 mg L−1 DNA was not affected by 8000-fold excesses Na+, 5000-fold excesses K+,3000-fold excesses of Ca2+, 2000-fold excesses of Mg2+,1000-fold excesses Zn 2+ , Al 3+ , lysine, ascorbic acid and uric acid. 500-fold excesses of Ag+, Hg2+ and Fe3+, and 300-fold excesses of l-cysteine and glutathione could significantly quench the RTP of MPA-capped Mn-doped ZnS QDs. Fortunately, the concentration of metal ions, l-cysteine and glutathione are much higher than present in samples, the interferences from biological fluids can be simply alleviated by diluting the samples. Sample Analysis Further experiment was performed to validate whether the Mn-doped ZnS QDs/RF nanohybrids can be used to determine DNA content. The standard addition recoveries on urine samples with DNA addition were 97 % to 103 % (Table S1). Pretreatment was not needed in all of the samples.

Conclusion The use of the developed RF-modulated Mn-doped ZnS QDs turn-on RTP sensor for quantitative detection of DNA was also demonstrated. The convenient method was applied to biological fluids with satisfactory results. This methodology could be developed for other biological molecules by replacing MPA or RF with high-recognition or selective functional groups for target analytes. Acknowledgments This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education, China (20121404110001), the Fund from Shanxi Province Chemical Advantage of Key Discipline Construction Projects, China (912019) and the Fund from Shanxi Province Postgraduate Innovation Project, China (104075).

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