Colloids and Surfaces B: Biointerfaces 117 (2014) 240–247

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

A sensitive quantum dots-based “OFF-ON” fluorescent sensor for ruthenium anticancer drugs and ctDNA Shan Huang a,∗ , Fawei Zhu a , Hangna Qiu a , Qi Xiao a,∗∗ , Quan Zhou a , Wei Su a,∗ ∗ ∗ , Baoqing Hu b a b

College of Chemistry and Life Science, Guangxi Teachers Education University, Nanning 530001, PR China Key Laboratory of Beibu Gulf Environment Change and Resources Utilization (Guangxi Teachers Education University), Ministry of Education, China

a r t i c l e

i n f o

Article history: Received 10 November 2013 Received in revised form 17 February 2014 Accepted 19 February 2014 Available online 28 February 2014 Keywords: CdTe quantum dots ctDNA fluorescent sensor photoinduced electron transfer ruthenium anticancer drugs

a b s t r a c t In this contribution, a simple and sensitive fluorescent sensor for the determination of both the three ruthenium anticancer drugs (1 to 3) and calf thymus DNA (ctDNA) was established based on the CdTe quantum dots (QDs) fluorescence “OFF-ON” mode. Under the experimental conditions, the fluorescence of CdTe QDs can be effectively quenched by ruthenium anticancer drugs because of the surface binding of these drugs on CdTe QDs and the subsequent photoinduced electron transfer (PET) process from CdTe QDs to ruthenium anticancer drugs, which render the system into fluorescence “OFF” status. The system can then be “ON” after the addition of ctDNA which brought the restoration of CdTe QDs fluorescence intensity, since ruthenium anticancer drugs broke away from the surface of CdTe QDs and inserted into double helix structure of ctDNA. The fluorescence quenching effect of the CdTe QDs-ruthenium anticancer drugs systems was mainly concentration dependent, which could be used to detect three ruthenium anticancer drugs. The limits of detection were 5.5 × 10−8 M for ruthenium anticancer drug 1, 7.0 × 10−8 M for ruthenium anticancer drug 2, and 7.9× 10−8 M for ruthenium anticancer drug 3, respectively. The relative restored fluorescence intensity was directly proportional to the concentration of ctDNA in the range of 1.0 × 10−8 M ∼ 3.0 × 10−7 M, with a correlation coefficient (R) of 0.9983 and a limit of detection of 1.1 × 10−9 M. The relative standard deviation (RSD) for 1.5 × 10−7 M ctDNA was 1.5% (n = 5). There was almost no interference to some common chemical compounds, nucleotides, amino acids, and proteins. The proposed method was applied to the determination of ctDNA in three synthetic samples with satisfactory results. The possible reaction mechanism of CdTe QDs fluorescence “OFF-ON” was further investigated. This simple and sensitive approach possessed some potential applications in the investigation of interaction between drug molecules and DNA. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In the past two decades, the water-soluble semiconductor quantum dots (QDs) have attracted widespread attention as novel fluorescence indicator in diverse research areas, such as analysis, sensing, and biodetection regions [1–5]. The confinement of the excited electron and the holes in QDs generates the specific optical and electronic properties that are dramatically different from those

∗ Corresponding author at: College of Chemistry and Life Science, Guangxi Teachers Education University, Nanning 530001, PR China Tel.: +86 771 3908065; fax: +86 771 3908308. ∗∗ Corresponding author. ∗ ∗ ∗ Corresponding author. E-mail addresses: [email protected] (S. Huang), [email protected] (Q. Xiao), [email protected] (W. Su). http://dx.doi.org/10.1016/j.colsurfb.2014.02.031 0927-7765/© 2014 Elsevier B.V. All rights reserved.

in bulk semiconductors [6]. Compared to traditional organic dyes and fluorescent proteins, QDs have some unique photophysical properties, for example, the broad/continuous excitation spectrum and narrow/symmetric emission spectrum, high photobleaching threshold, high emission quantum yield, and so on [7–9]. These advantages make QDs to be excellent probes for some chemical and biological assay [10–12]. Some metal ions, small molecules, and biomacromolecules could be detected by using QDs as fluorescent probe based on fluorescence quenching or fluorescence enhancement phenomenon [13–19]. Nie and coworkers have reported an ultrasensitive and selective detection of Cu2+ with the fluorescence enhancement of CdSe QDs [13]. Zhou et al. have developed a novel luminescence sensing system for Sudan dye detection, based on the fluorescence quenching of oleic acid-functionalized Mn-ZnS QDs [15]. He’s group further used the fluorescence increment phenomenon of CdSe/ZnS QDs to detect L-cysteine [17]. Ding et al. have explored specific and selective detection approach for

S. Huang et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 240–247

Fig. 1. A) The chemical structures of ruthenium anticancer drugs 1 to 3 after the detachment of the chloride ions. B) The schematic illustration of the principle of ruthenium anticancer drugs detection and ctDNA determination using the CdTe QDs-based “OFF-ON” fluorescent sensor.

bovine serum albumin (BSA) by fluorescence enhancement of Mnmodified CdTe QDs [19]. However, the fluorescence quenching and the fluorescence enhancement of QDs all belong to the unidirectional fluorescence variations of QDs which can be easily affected by some foreign substances. In order to expand the applications of QDs probe, new methods should be explored to increase the selectivity of QDs probe irrespective of any situation. Fortunately, a novel and sensitive fluorescence “OFF-ON” sensor based on QDs had been explored recently [20–28]. In our previous work, through the specific and strong interactions between streptavidin and biotin or between single-stranded DNA and the complementary single-stranded DNA, the fluorescence of QDs were quenched efficiently by fluorescent dye due to the fluorescence resonance energy transfer (FRET). The fluorescence of QDs could be recovered partially after the addition of specific nuclease or targeted DNA because of the disturbance of FRET effects from QDs to fluorescent dye [20,21]. Since none of the common foreign substances could affect the FRET efficiency, this QDs-based “OFF-ON” fluorescent sensor ensured excellent selectivity in specific nuclease and targeted DNA detection. In addition, this fluorescence “OFFON” mode had attracted widespread interests and had realized simple and sensitive determination of different substances, such as Hg2+ , CN− , mitoxantrone, DNA, nuclease, and viruses [22–28]. Furthermore, this novel mode could also be applied for the investigation of the interactions between some functional drugs and their targets [29,30]. Therefore, this new fluorescence “OFF-ON” sensor should be further studied to exploit their potential application in biochemical and biomedical detections. As one kind of the organometallic compounds, ruthenium complexes have attracted great interests due to their specific antibacterial, antitumor, and anticancer activity [31–34]. The bioactivity effects of these ruthenium complexes are exerted by the inhibition of DNA or RNA replication and amplification after their intercalation into the double helix of DNA inside the cancer cells and then combination with the base pairs, which lead to the death of the cancer cells [35,36]. The investigation of the binding interaction of some bioactive ruthenium complex drugs to their target DNA has been an active area of research, which could make us better understand the mechanism of their antibacterial, antitumor, and anticancer activity, together with the directional design and the efficient synthesis of new functional drugs [37–39]. Until now, the binding interaction between different ruthenium complex drugs and their target DNA has already been investigated by many approaches [40–48]. However, to our knowledge, the application of QDs as the fluorescent sensor to test the ruthenium anticancer drugs-DNA interaction has rarely been reported till now. Herein, we establish a simple and sensitive QDs-based “OFFON” fluorescent sensor by utilizing ruthenium anticancer drug as

241

both the quencher to QDs and the intercalating agent to calf thymus DNA (ctDNA) (Fig. 1). It is well known that the possible quenching mechanism of ruthenium complex to QDs is mainly photoinduced electron transfer (PET) process [49,50]. As shown in Fig. 1, based on the electrostatic interactions, the negatively-charged CdTe QDs could be combined with the positively-charged ruthenium anticancer drugs after the detachment of the chloride ions. Because of the surface combination of ruthenium anticancer drugs on CdTe QDs, PET could occur easily between CdTe QDs and ruthenium anticancer drugs. Meanwhile, ruthenium (II), which is the central ion of the ruthenium anticancer drugs, has partially filled d-orbitals and can be used as electron acceptor. The ultrafast PET from CdTe QDs to ruthenium anticancer drugs prevented the normal recombination of electron and hole in CdTe QDs, which resulted in the fluorescence “OFF” status of CdTe QDs. After the addition of ctDNA, the positively-charged ruthenium anticancer drugs could combine with the negatively-charged ctDNA through electrostatic forces and insert into the double helix structure of ctDNA [35,37,38]. Since the mutual repulsion between both the negatively-charged ctDNA and the negatively-charged CdTe QDs existed, the ctDNA-ruthenium anticancer drug complexes could break away from the surface of CdTe QDs, and this interrupted the electron transfer from CdTe QDs to ruthenium anticancer drugs and rendered the fluorescence “ON” status of CdTe QDs. This QDs-based “OFF-ON” fluorescent sensor could be used to detect both ruthenium anticancer drugs and ctDNA with the properties of simplicity, rapidity, and sensitivity. 2. Experimental 2.1. Reagents Tellurium (powder, 200 meshes, 99.8%), sodium borohydride (NaBH4 , 99.8%), CdCl2 •H2 O (99.99%), and N-acetyl-L-cysteine (NAC, ≥ 99%) were purchased from Sigma (St. Louis, MO, USA). ctDNA, nucleotides, and amino acids were obtained from Sinopharm Chemical Reagent Factory (Shanghai, China). Three ruthenium anticancer drugs (1 to 3) were synthesized according to the reference reported previously [51]. BSA, papain, lysozyme, and pepsin were purchased from Beijing Huamei Bioscience Technology (Beijing, China). All other reagents were of analytical-reagent grade and used as received without further purification. Ultrapure water with a resistivity of 18.2 M cm was produced by passing through a RiOs 8 unit followed by a Millipore-Q Academic purification set (Millipore, Bedford, MA, USA) and used throughout the experiments. 2.2. Apparatus The absorption spectra were measured on a TU-1901 UVvis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). All fluorescence spectra and intensities were recorded with a Perkin-Elmer Model LS-55 luminescence spectrometer (PerkinElmer, Waltham, MA, USA) equipped with a 20KW xenon discharge lamp as light source. Quartz cells (1 cm path-length) were used for all measurements. The time-resolved fluorescence decay traces were recorded with a Fluorolog-3 system (Horiba Jobin Yvon, France) by using an excitation wavelength of 374 nm. All pH measurements were made with a basic pH meter PB-10 (Sartorius Scientific Instruments Co., Ltd., Beijing, China). 2.3. Preparations of CdTe QDs The CdTe QDs were synthesized according to the method described previously [52,53]. In a typical synthesis, 0.2 mmol tellurium powder and 1.0 mmol NaBH4 were put into a 25 mL two-necked flask equipped with a constant pressure funnel which contained 5.0 mL ultrapure water. Air in this system was pumped

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off and then replaced with N2 . The reaction mixture was heated to 80 ◦ C and reacted at this temperature for 30 min under N2 flow protection until it became dark red. The obtained NaHTe solution was stored under N2 protection for further use at room temperature. Then, 0.2 mmol CdCl2 and 0.34 mmol NAC solution were mixed in a 40 mL solution. The pH of the mixture was adjusted to 12.0 by adding 1.0 M NaOH solution dropwise under stirring. The mixture was transferred into a three-necked flask. Air in the system was pumped off and replaced with N2 . Under stirring, 1 mL NaHTe solution (0.04 mmol) was added into the Cd precursor solution by syringe at room temperature. The molar ratio of [Cd]: [Te]: [NAC] was fixed at 1.0: 0.2: 1.7. Then the mixture was heated to 100 ◦ C and reacted at this temperature for 8 min. The heat was immediately removed. The CdTe QDs samples were taken when the temperature cooled down to room temperature. To remove the excess NAC-Cd complexes at the end of the synthesis, cold 2-propanol was added to the reaction mixture to precipitate CdTe QDs. The as-prepared precipitate was dried overnight under vacuum at 30 ◦ C and then redispersed in ultrapure water and stored in refrigerator for further experiments. The solution concentrations were estimated from the absorption spectra using the molar absorptivity at the first absorption maximum for CdTe QDs with this diameter reported by Peng and coworkers [54]. 2.4. Preparation of synthesis samples For the synthetic samples detection, three samples were prepared by mixing the standard solutions of different solution with different concentrations in the reaction system. The concentrations of KNO3 , NaNO3 , L-Cysteine, L-Lysine, L-Glycine, guanine, and BSA in sample 1 were 5.0 × 10−5 M, 2.0 × 10−4 M, 3.0 × 10−6 M, 5.0 × 10−6 M, 6.0 × 10−6 M, 6.0 × 10−6 M, and 1.0 × 10−6 M, respectively. The concentrations of Al(NO3 )3 , L-Glutamine, L-Tyrosine, L-Methionine, L-Alanine, adenine, cytosine, and papain in sample 2 were 1.0 × 10−5 M, 5.0 × 10−6 M, 5.0 × 10−6 M, 3.0 × 10−6 M, 5.0 × 10−6 M, 5.0 × 10−6 M, 5.0 × 10−6 M, and 1.0 × 10−6 M, respectively. Sample 3 contained 2.0 × 10−5 M CuSO4 , 4.0 × 10−6 M L-Phenylalanine, 8.0 × 10−6 M L-Leucine, 6.0 × 10−6 M LHistidine, 1.0 × 10−5 M thymine, 2.0 × 10−7 M lysozyme, and 2.0 × 10−6 M pepsin. Different amounts of ctDNA standard solution were added into the reaction system and the final concentration of ctDNA was 1.5 × 10−7 M in sample 1, 3.0 × 10−7 M in sample 2, and 5.0 × 10−7 M in sample 3, respectively. The recovery of ctDNA in three synthetic samples was examined by the proposed method. 2.5. Ruthenium anticancer drugs and ctDNA detection For the three ruthenium anticancer drugs determination, 50 ␮L 1.0 × 10−5 M of CdTe QDs, 1.5 mL Tris-HCl (pH 7.4), and the appropriate aliquot of ruthenium anticancer drug solution were transferred orderly into a 5 mL eppendorf tube. The mixture was stirred thoroughly and finally diluted to 3 mL with ultrapure water. After 30 min incubation at room temperature, the fluorescence spectra were measured for the quantitative analysis of the three ruthenium anticancer drugs. For ctDNA detection, 50 ␮L 1.0 × 10−5 M of CdTe QDs, 1.5 mL Tris-HCl (pH 7.4), and the appropriate aliquot of ruthenium anticancer drug 3 solution were transferred orderly into a 5 mL eppendorf tube and the mixture was stirred thoroughly. After incubation for 30 min, the appropriate aliquot of ctDNA were added into the mixture and finally diluted to 3 mL with ultrapure water. After additional incubation of 30 min at room temperature, the fluorescence spectra were measured for the quantitative analysis of ctDNA. When samples were determined, ctDNA standard

Fig. 2. The normalized UV-vis absorption (a) and fluorescence (b) spectra of CdTe QDs at room temperature. Insert: Photograph of emission color of CdTe QDs under the radiation of UV lamp.

solution was substituted by the prepared sample solution described in Section 2.4. The fluorescence spectra were recorded at excitation wavelength of 388 nm and the band-slits of both excitation and emission were set as 10.0 nm and 5.0 nm, respectively. The fluorescence spectra were recorded from 450 nm to 650 nm, the fluorescence intensity of CdTe QDs at 525 nm was used for quantitative analysis of both ruthenium anticancer drugs and ctDNA. 3. Results and Discussion 3.1. Properties of CdTe QDs The representative normalized UV-vis absorption (a) and fluorescence (b) spectra of CdTe QDs at room temperature are shown in Fig. 2. It could be seen from Fig. 2a that the first absorption maximum wavelength of CdTe QDs was at 495 nm. The diameter of these CdTe QDs could be calculated from the first absorption maximum wavelength [54], and the results indicated that the diameter of these CdTe QDs were about 2.1 nm. It could be seen from Fig. 2b that these CdTe QDs exhibited an obvious, symmetrical fluorescence emission spectrum without a tail on the right-hand side and the emission maximum wavelength of these CdTe QDs was at 525 nm with an excitation wavelength of 388 nm. The line width of the fluorescence spectrum of CdTe QDs was narrow, indicating that the as-prepared CdTe QDs were nearly monodisperse and homogeneous. The fluorescence quantum yield (QY) of CdTe QDs was determined according to the reported method [55] by using fluorescein as fluorescence standard (QY = 79% in 0.1 M NaOH solution). The experimental results indicated that the fluorescence QY of CdTe QDs was about 26%. 3.2. Characterization of QDs-based fluorescence “OFF-ON” mode The CdTe QDs fluorescence “OFF status” was based on the effective fluorescence quenching of CdTe QDs by ruthenium anticancer drugs. The normalized fluorescence spectra of CdTe QDs with different kinds of ruthenium anticancer drugs were shown in Fig. 3A. It could be seen from Fig. 3A that the fluorescence of CdTe QDs was seriously quenched after the addition of the three ruthenium anticancer drugs. Furthermore, 2.0 × 10−5 M of drug 1, 2.5 × 10−5 M of drug 2, and 5.0 × 10−5 M of drug 3 presented a quenching effect of 59%, 37%, and 55%, respectively. Since 1.5 × 10−6 M, 1.0 × 10−5 M, and 5.0 × 10−5 M of drug 3 presented a quenching effect of 30%, 43%, and 55%, respectively, it could be deduced easily that the

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Fig. 3. A) The normalized fluorescence spectra of 1.7 × 10−7 M CdTe QDs (a) at different concentrations of ruthenium anticancer drugs in Tris-HCl buffer. The concentrations of different ruthenium anticancer drugs were: (b) 2.0 × 10−5 M for drug 1; (c) 2.5 × 10−5 M for drug 2; (d) 1.5 × 10−6 M for drug 3; (e) 1.0 × 10−5 M for drug 3; (f) 5.0 × 10−5 M for drug 3. B) The normalized fluorescence spectra of CdTe QDs alone (a), CdTe QDs with 1.0 × 10−6 M ctDNA (b), and CdTe QDs-ruthenium anticancer drug 3 systems (c) with different concentrations of ctDNA. CdTe QDs: 1.7 × 10−7 M; ruthenium anticancer drug 3: 1.5 × 10−4 M; ctDNA: 6.0 × 10−7 M (d), and 1.0 × 10−6 M (e).

fluorescence quenching effect of the CdTe QDs-ruthenium anticancer drug systems was mainly concentration dependent, which could be applied to the determination of ruthenium anticancer drugs. Furthermore, the fluorescence peaks of the CdTe QDsruthenium anticancer drug systems were constant at 525 nm without any shift, demonstrating the inferred PET mechanism between CdTe QDs and ruthenium anticancer drugs. The fluorescence of CdTe QDs-ruthenium anticancer drug systems could be “ON” after the addition of ctDNA. The normalized fluorescence spectra of both CdTe QDs with ctDNA and CdTe QDsruthenium anticancer drug 3 systems with different concentration of ctDNA are shown in Fig. 3B. According to Fig. 3B, the fluorescence intensity of CdTe QDs was almost not influenced by 1.0 × 10−6 M of ctDNA. However, the fluorescence intensity of CdTe QDsruthenium anticancer drug 3 systems was restored gradually with the increasing of the concentration of ctDNA. 6.0 × 10−7 M and 1.0 × 10−6 M of ctDNA presented a restoring effect of 41% and 63%, respectively, so the fluorescence restoring effect was mainly ctDNA concentration dependent and the fluorescence restoration approach could be used for the sensitive detection of ctDNA.

3.3. Detection of ruthenium anticancer drugs The influence of the reaction time on the fluorescence quenching of CdTe QDs by ruthenium anticancer drugs was studied. Preliminary experiments demonstrated that the fluorescence quenching of CdTe QDs by ruthenium anticancer drugs were finished within 10 min and the fluorescence signals could remain constant for more than 90 min, which indicated that the CdTe QDs-ruthenium anticancer drugs systems exhibited good stability (Fig. S1). Therefore, the fluorescence intensities of CdTe QDs-ruthenium anticancer drug systems were recorded after the addition of ruthenium anticancer drugs for 30 min. Since the pH value played a quite important role in the interaction between QDs and other molecules [17,56], the effect of different pH values on the fluorescence intensity reflecting the interaction of CdTe QDs with ruthenium anticancer drugs was investigated from pH 6.6 to 10.0 (Fig. S2). It was found that the fluorescence intensity of CdTe QDs increased gradually with the increment in pH value from 6.6 to 7.4. When pH value was higher than 7.4, the fluorescence intensity of CdTe QDs decreased dramatically. The variation trends of the fluorescence intensities of CdTe QDs-ruthenium anticancer drugs systems were consistent with that of the CdTe QDs alone. The maximum changes of fluorescence intensities occurred when pH value was 7.4. Therefore, 0.01 M pH 7.4 Tris-HCl buffer solution was chosen for further

experiments. Simultaneously, the impact of the volume of TrisHCl buffer solution on the reaction was investigated. The results indicated that the maximum changes of fluorescence intensities occurred when the volume of Tris-HCl buffer solution was 1.5 mL (Fig. S3), so 1.5 mL Tris-HCl buffer solution was selected as the reaction medium. The influence of CdTe QDs volume on the fluorescence quenching efficiency of CdTe QDs-ruthenium anticancer drugs systems was also tested. The previous experimental results showed that the quench efficiency of the fluorescence intensity of CdTe QDs by ruthenium anticancer drugs increased obviously when the volume of CdTe QDs was in the range of 10 ∼ 50 ␮L (Fig. S4). When the volume of CdTe QDs was higher than 50 ␮L, the quench efficiency decreased gradually. Higher CdTe QDs concentration would disturb the electrostatic balance of the electrostatically stabilized CdTe QDs and cause the aggregation of CdTe QDs themselves, which might affect the PET process from CdTe QDs to ruthenium anticancer drugs. Since the maximum changes of fluorescence intensities appeared at 50 ␮L CdTe QDs, 50 ␮L CdTe QDs was chosen in this study. Furthermore, the effects of ionic intensity on the systems were also studied by choosing NaCl as the ionic strength adjustor (Fig. S5). Higher concentration of Na+ or Cl− had competitive influence on the interaction between CdTe QDs and ruthenium anticancer drugs as reported before [29]. Therefore, NaCl was not added in the system and the reaction was under a lower ionic strength condition. Under the optimum experimental conditions, the fluorescence intensity quenching of CdTe QDs was proportional to the concentration of ruthenium anticancer drugs 1 to 3 in certain ranges, which could be described by the well-known Stern–Volmer equation [57,58]:

I0 = 1 + KSV [Q ], I where I0 and I are the fluorescence intensities in the absence and presence of the quencher, respectively, KSV is the Stern–Volmer quenching constant, and [Q] is the concentration of the quencher, respectively. The linear regression equation, linear ranges, detection limits, and correlation coefficients were obtained and listed in Table 1. The relative standard deviation (RSD) for 1.6 × 10−6 M drug 1, 3.0 × 10−6 M drug 2, and 2.0 × 10−6 M drug 3 were 2.1%, 1.5%, and 1.8% (n = 5), respectively. According to the results, the proposed methods can be used for the sensitive determination of ruthenium anticancer drugs.

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Table 1 The linear regression equation, linear ranges, detection limits, and correlation coefficients. Drugs

Linear regression equation

Linear ranges

Detection limits

Correlation coefficients

1 2 3

I0 /I = 1 + 0.04216 [Q] I0 /I = 1 + 0.02147 [Q] I0 /I = 1 + 0.1019 [Q]

3.0 × 10−7 M ∼ 1.0 × 10−5 M 8.0 × 10−7 M ∼ 1.3 × 10−5 M 3.0 × 10−7 M ∼ 5.0 × 10−6 M

5.5 × 10−8 M 7.0 × 10−8 M 7.9 × 10−8 M

0.9997 0.9992 0.9998

Table 3 Determination of ctDNA in three synthetic samples (n = 5)a .

Fig. 4. The fluorescence spectra of CdTe QDs alone and CdTe QDs-ruthenium anticancer drug 3 systems in different concentrations of ctDNA: (a) 0 M; (b) 5.0 × 10−8 M; (c) 2.0 × 10−7 M; (d) 4.0 × 10−7 M; (e) 6.0 × 10−7 M; (f) 8.0 × 10−7 M; (g) 1.0 × 10−6 M; (h) 1.5 × 10−6 M. The insert was the linear relationship between the I – I0 and ctDNA concentration in the range of 1.0 × 10−8 M to 3.0 × 10−7 M with a correlation coefficient of 0.9983. The relative standard deviation (RSD) for ctDNA in this range were 2.0% for 1.0 × 10−8 M, 1.6% for 4.0 × 10−8 M, 1.7% for 7.0 × 10−8 M, 1.9% for 1.2 × 10−7 M, 2.2% for 1.8 × 10−7 M, and 3.4% for 3.0 × 10−7 M, respectively (n = 5). CdTe QDs: 1.7 × 10−7 M; ruthenium anticancer drug 3: 5.0 × 10−4 M.

3.4. Determination of ctDNA The influence of the reaction time on the fluorescence “ON” of CdTe QDs-ruthenium anticancer drugs systems by adding ctDNA was studied. Preliminary experiments demonstrated that the fluorescence restoration of CdTe QDs-ruthenium anticancer drugs systems by adding ctDNA were finished within 15 min and lasted for more than 120 min, which indicated that the CdTe QDsruthenium anticancer drugs systems exhibited higher stability. Therefore, the fluorescence intensities of CdTe QDs-ruthenium anticancer drugs systems with ctDNA were measured after adding ctDNA for 30 min. Under the optimum conditions, the fluorescence intensity restoration of CdTe QDs-ruthenium anticancer drug 3 systems was proportional to the concentration of ctDNA in the range of 1.0 × 10−8 M ∼ 3.0 × 10−7 M with a correlation coefficient of 0.9983. The relative standard deviation (RSD) for 1.5 × 10−7 M ctDNA was 1.5%

Synthetic samples

Taken (× 10−8 M)

Found (× 10−8 M)

Recovery (%)

RSD (%)

Sample 1b Sample 2c Sample 3d

15 30 50

15.6 29.2 51.3

104.0 97.3 102.6

1.3 2.1 2.0

a CdTe QDs: 1.7 × 10−7 M; Ruthenium anticancer drug 3: 2.0 × 10−6 M; ctDNA: 3.5 × 10−7 M. b 5.0 × 10−5 M KNO3 , 2.0 × 10−4 M NaNO3 , 3.0 × 10−6 M L-Cysteine, 5.0 × 10−6 M L-Lysine, 6.0 × 10−6 M L-Glycine, 6.0 × 10−6 M guanine, and 1.0 × 10−6 M BSA. c 1.0 × 10−5 M Al(NO3 )3 , 5.0 × 10−6 M L-Glutamine, 5.0 × 10−6 M L-Tyrosine, 3.0 × 10−6 M L-Methionine, 5.0 × 10−6 M L-Alanine, 5.0 × 10−6 M adenine, 5.0 × 10−6 M cytosine, and 1.0 × 10−6 M papain. d 2.0 × 10−5 M CuSO4 , 4.0 × 10−6 M L-Phenylalanine, 8.0 × 10−6 M L-Leucine, 6.0 × 10−6 M L-Histidine, 1.0 × 10−5 M thymine, 2.0 × 10−7 M lysozyme, and 2.0 × 10−6 M pepsin.

(n = 5). The fluorescence spectra of CdTe QDs-ruthenium anticancer drug 3 systems at different concentrations of ctDNA between 1.0 × 10−8 M and 3.0 × 10−7 M were shown in Fig. 4. The linear relationship between the change of fluorescence intensity of CdTe QDs and ctDNA concentration in that range was also shown in the insert of Fig. 4. The linear regression equation was I – I0 = 2.79 × [ctDNA]. Based on the three times standard deviation of 12 measurements of CdTe QDs-ruthenium anticancer drug 3 solution without ctDNA, the limit of detection for ctDNA was up to 1.1 × 10−9 M. 3.5. Effect of foreign substances and sample determination In order to verify the applicability of this approach for ctDNA determinations in biological samples, the influence of some common chemical compounds, nucleotides, amino acids, and proteins were investigated and the results are shown in Table 2. If the coexisting substances caused a relative error of less than ± 5% in the fluorescence intensity of the system, they were considered to have no interference with the ctDNA determination. It could be seen from Table 2 that some common chemical compounds, 4 nucleotides, and some amino acids had almost no distinct effect on the determination of 3.5 × 10−7 M ctDNA. Furthermore, 3.0 × 10−6 M BSA, 5.0 × 10−6 M papain, 5.0 × 10−7 M lysozyme, and 5.0 × 10−6 M pepsin did not affect the detection of

Table 2 Effect of coexisting foreign substances (n = 5)a . Foreign substances

Concentration coexisting (× 10−6 M)

Change of FL Intensity (%)

R. S. D. (%)

Foreign substances

Concentration coexisting (× 10−6 M)

Change of FL Intensity (%)

R. S. D. (%)

KNO3 NaNO3 Al(NO3 )3 CuSO4 BSA Papain Pepsin Lysozyme adenine

100 800 40 40 3 5 5 0.5 20

− 3.1 − 2.8 + 1.2 − 1.9 − 2.5 − 3.8 + 2.2 + 2.4 − 3.3

1.1 0.7 0.6 2.1 0.9 1.3 1.2 1.5 2.2

20 10 10 20 15 10 14 20 10

− 1.4 − 2.1 + 4.7 − 3.6 − 4.3 + 4.1 − 3.8 + 2.7 − 3.3

0.6 0.9 1.3 1.5 2.0 2.1 2.4 1.3 1.4

cytosine thymine

20 20

− 2.6 − 3.1

1.0 2.3

guanine L-Cysteine L-Lysine L-Glycine L-Glutamine L-Tyrosine L-Methionine L-Alanine LPhenylalanine L-Leucine L-Histidine

20 18

− 3.4 − 2.8

1.5 1.8

a

CdTe QDs: 1.7 × 10−7 M; Ruthenium anticancer drug 3: 2.0 × 10−6 M; ctDNA: 3.5 × 10−7 M.

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Fig. 5. A) The UV-vis absorption spectra of CdTe QDs alone, ruthenium anticancer drug 3, ctDNA alone, and CdTe QDs-ruthenium anticancer drug 3 system; the difference absorption spectra between CdTe QDs-ruthenium anticancer drug 3 system and ruthenium anticancer drug 3, and between ctDNA-ruthenium anticancer drug 3 system and ruthenium anticancer drug 3, respectively. B) The fluorescence decay traces of CdTe QDs alone (a), CdTe QDs-ruthenium anticancer drug 3 system (b), and CdTe QDsruthenium anticancer drug 3 systems reacted with 8.0 × 10−7 M ctDNA (c) in solution. All measurements were made at em = 525 nm. The concentrations of CdTe QDs and ruthenium anticancer drug 3 were 1.7 × 10−7 M and 5.0 × 10−5 M, respectively.

3.5 × 10−7 M ctDNA in the given conditions. The data revealed that the proposed method might be applied to the detection of ctDNA in biological samples. To confirm the feasibility, the present method was also applied to the determination of ctDNA in three synthetic samples which contained 4 common chemical compounds, 10 amino acids, 4 nucleotides, and 4 proteins. As indicated in Table 3, the values found for the three synthetic samples were identical with the expected values and the recoveries were from 97.3% to 104.0%, which indicated the suitability of ctDNA determination in the mixture of these substances. 3.6. The mechanism of fluorescence “OFF-ON” of CdTe QDs It is well known that the mechanism of fluorescence quenching usually includes the dynamic quenching (collisional process) and the static quenching (ground state complex formation), which can be distinguished by careful examination of the UV-vis absorption spectra of the fluorophore [57,59]. In the dynamic quenching, charge transfer often occurs and fluorescence is subsequently quenched when the electron acceptor collides with the excited fluorophore, so no variations in the absorption spectra of the fluorophore will be expected. However, in the static quenching, the formation of ground state complex will perturb the absorption spectra of the fluorophore. So, the UV-vis absorption spectra of CdTe QDs, ruthenium anticancer drug 3, and CdTe QDs-ruthenium anticancer drug 3 systems were recorded to confirm the probable fluorescence quenching mechanism of CdTe QDs-ruthenium anticancer drug 3 systems. Furthermore, the UV-vis absorption spectrum of ctDNA and the difference absorption spectra between ctDNA-ruthenium anticancer drug 3 systems and ruthenium anticancer drug 3 were also used to investigate the intercalation interaction between ctDNA and ruthenium anticancer drug 3. As shown in Fig. 5A, the pure CdTe QDs showed strong absorption at wavelengths < 450 nm and relatively weak absorption in the visible region. In the absorption spectrum of pure ruthenium anticancer drug 3, there are two strong peaks at 225 nm and 321 nm. In order to study the change of absorption spectrum intensity on the interaction between CdTe QDs and ruthenium anticancer drug 3, the difference absorption spectrum between CdTe QDs-ruthenium anticancer drug 3 systems and ruthenium anticancer drug 3 is also shown (Fig. 5A). It was clear that the UV-vis absorption spectrum of CdTe QDs and the difference absorption spectrum between CdTe QDs-ruthenium anticancer drug 3 systems and ruthenium anticancer drug 3 could be superposed

perfectly within the experimental error, implying that no conjugation or particle-size variation occurred in the process of the fluorescence quenching. In addition, the absorption spectrum of ctDNA and the difference absorption spectrum between ctDNAruthenium anticancer drug 3 systems and ruthenium anticancer drug 3 were totally different, indicating that the intercalation interaction occurred between ctDNA and ruthenium anticancer drug 3. These results conformed to the dynamic quenching mechanism of CdTe QDs by ruthenium anticancer drug 3 that resulted from the collisional process between CdTe QDs and ruthenium anticancer drug 3. Moreover, the dynamic quenching and the static quenching can also be distinguished directly by testing the fluorescence lifetime of the fluorophore [57]. The dynamic quenching can affect the excited states of the fluorophore and obvious variations will be expected in the fluorescence lifetime of the fluorophore, while the static quenching will not cause the perturbation of the fluorescence lifetime of the fluorophore [56,57]. Therefore, the time-resolved fluorescence spectra of CdTe QDs and CdTe QDs-ruthenium anticancer drug 3 systems with or without ctDNA were measured to investigate the photophysical properties of these systems. The fluorescence decay curves of these systems were well fitted with biexponential equation (Insert in Fig. 5B). If the intensity decays are multiexponential, it is important to use an average decay time which is proportional to the steady state intensity and the average values are given by the sum of the bi  i products [21,56,60]. As shown in Fig. 5B, CdTe QDs exhibited two fluorescence decay components,  1 5.97 ± 0.36 ns (40.63%) and  2 16.76 ± 0.36 ns (59.37%). The corresponding average decay time of CdTe QDs was about 12.38 ns. When ruthenium anticancer drug 3 was added into the solution, two fluorescence decay components of CdTe QDs were shortened to  1 2.17 ± 0.46 ns (26.49%) and  2 8.78 ± 0.48 ns (73.51%). The average decay time of CdTe QDs was also decreased to around 7.03 ns. These results further indicated the dynamic quenching of CdTe QDs by ruthenium anticancer drug 3 and also confirmed the occurrence of PET process between CdTe QDs and ruthenium anticancer drugs (Fig. 1). After the addition of ctDNA into the CdTe QDs-ruthenium anticancer drug 3 systems, two fluorescence decay components of CdTe QDs were increased to  1 3.94 ± 0.24 ns (41.64%) and  2 12.76 ± 0.41 ns (58.36%). The CdTe QDs also recovered their original average decay time partially, 9.09 ns. These results also indicated the incomplete intercalation interaction between ctDNA and ruthenium anticancer drug 3 (Fig. 1), which resulted in the restoration of the fluorescence of CdTe QDs partly.

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4. Conclusions A simple and sensitive CdTe QDs-based “OFF-ON” fluorescent sensor was established to detect both ruthenium anticancer drugs and ctDNA in this paper. The primary advantage of this method is its simplicity, rapidity, and sensitivity. The fluorescence of CdTe QDs could be quenched by ruthenium anticancer drugs through PET process, which made the fluorescence of CdTe QDs into “OFF” status. However, the fluorescence of CdTe QDs could be “ON” after the addition of ctDNA which could take ruthenium anticancer drugs away from CdTe QDs. Under the optimum conditions, the limits of detection for ruthenium anticancer drug 1, ruthenium anticancer drug 2, ruthenium anticancer drug 3, and ctDNA were 5.5 × 10−8 M, 7.0 × 10−8 M, 7.9 × 10−8 M, and 1.1 × 10−9 M, respectively. Some common chemical compounds, nucleotides, amino acids, and proteins could not affect the ctDNA determination. The method presented here has been applied to the determination of ctDNA in synthetic samples successfully. The possible mechanism among them was also investigated by UV-vis spectroscopy and time-resolved fluorescence spectroscopy. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21203035, 21261005), the Guangxi Natural Science Foundation (2013GXNSFCA019005, 2013GXNSFBA019029), the Scientific Research Foundation of Guangxi Provincial Education Department (2013YB138), Ministry of Education, China and the 46th Scientific Research Foundation for the Returned Overseas Chinese Scholars (13XJA810001), Ministry of Education, China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.02.031. References [1] M. Bruchez, M. Moronne Jr., P. Gin, S. Weiss, A.P. Alivisatos, Semiconductor nanocrystals as fluorescent biological labels, Science 281 (1998) 2013. [2] I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Quantum dot bioconjugates for imaging, labelling and sensing, Nat. Mater. 4 (2005) 435. [3] R. Freeman, R. Gill, I. Shweky, M. Kotler, U. Banin, I. Willner, Biosensing and probing of intracellular metabolic pathways by NADH-sensitive quantum dots, Angew. Chem. Int. Ed. 48 (2009) 309. [4] R.Z. Liang, R. Tian, W.Y. Shi, Z.H. Liu, D.P. Yan, M. Wei, D.G. Evans, X. Duan, A temperature sensor based on CdTe quantum dots–layered double hydroxide ultrathin films via layer-by-layer assembly, Chem. Commun. 49 (2013) 969. [5] W.L. Sun, J.L. Yao, T.M. Yao, S. Shi, Label-free fluorescent DNA sensor for the detection of silver ions based on molecular light switch Ru complex and unmodified quantum dots, Analyst 138 (2013) 421. [6] C.J. Murphy, Optical sensing with quantum dots, Anal. Chem. 74 (2002) 520A. [7] V.S. Pribiag, S. Nadj-Perge, S.M. Frolov, J.W.G. van den Berg, I. van Weperen, S.R. Plissard, E.P.A.M. Bakkers, L.P. Kouwenhoven, Electrical control of single hole spins in nanowire quantum dots, Nat. Nanotechnol. 8 (2013) 170. [8] K. Boeneman Gemmill, J.R. Deschamps, J.B. Delehanty, K. Susumu, M.H. Stewart, R.H. Glaven, G.P. Anderson, E.R. Goldman, A.L. Huston, I.L. Medintz, Optimizing protein coordination to quantum dots with designer peptidyl linkers, Bioconjugate Chem. 24 (2013) 269. [9] E.A. Chekhovich, M.N. Makhonin, A.I. Tartakovskii, A. Yacoby, H. Bluhm, K.C. Nowack, L.M.K. Vandersypen, Nuclear spin effects in semiconductor quantum dots, Nat. Mater. 12 (2013) 494. [10] K.T. Yong, W.C. Law, R. Hu, L. Ye, L.W. Liu, M.T. Swihart, P.N. Prasad, Nanotoxicity assessment of quantum dots: from cellular to primate studies, Chem. Soc. Rev. 42 (2013) 1236. [11] M.G. Panthani, T.A. Khan, D.K. Reid, D.J. Hellebusch, M.R. Rasch, J.A. Maynard, B.A. Korgel, In vivo whole animal fluorescence imaging of a microparticle-based oral vaccine containing (CuInSex S2–x )/ZnS core/shell quantum dots, Nano Lett. 13 (2013) 4294. [12] G.W. Platt, F. Damin, M.J. Swann, I. Metton, G. Skorski, M. Cretich, M. Chiari, Allergen immobilisation and signal amplification by quantum dots for use in a biosensor assay of IgE in serum, Biosens. Bioelectron. 52 (2014) 82.

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A sensitive quantum dots-based "OFF-ON" fluorescent sensor for ruthenium anticancer drugs and ctDNA.

In this contribution, a simple and sensitive fluorescent sensor for the determination of both the three ruthenium anticancer drugs (1 to 3) and calf t...
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