Platinum(II)-Oligonucleotide Coordination Based Aptasensor for

Sep 22, 2015 - Wide use of platinum-based chemotherapeutic regimens for the treatment for carcinoma calls for a simple and selective detection of plat...
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Platinum(II)-Oligonucleotide Coordination Based Aptasensor for Simple and Selective Detection of Platinum Compounds Sheng Cai, Xueke Tian, Lianli Sun, Haihong Hu, Shirui Zheng, Huidi Jiang, Lushan Yu,* and Su Zeng* Laboratory of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China S Supporting Information *

ABSTRACT: Wide use of platinum-based chemotherapeutic regimens for the treatment for carcinoma calls for a simple and selective detection of platinum compound in biological samples. On the basis of the platinum(II)-base pair coordination, a novel type of aptameric platform for platinum detection has been introduced. This chemiluminescence (CL) aptasensor consists of a designed streptavidin (SA) aptamer sequence in which several base pairs were replaced by GG mismatches. Only in the presence of platinum, coordination occurs between the platinum and G-G base pairs as opposed to the hydrogen-bonded G-C base pairs, which leads to SA aptamer sequence activation, resulting in their binding to SA coated magnetic beads. These Pt-DNA coordination events were monitored by a simple and direct luminol-peroxide CL reaction through horseradish peroxidase (HRP) catalysis with a strong chemiluminescence emission. The validated ranges of quantification were 0.12−240 μM with a limit of detection of 60 nM and selectivity over other metal ions. This assay was also successfully used in urine sample determination. It will be a promising candidate for the detection of platinum in biomedical and environmental samples.

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applied for the detection of metal ions. By coupling to fluorescent, electrochemical, or colorimetric techniques, some simple detection platforms have been developed for lead(II)19−21 and mercury(II) ions,22−24 among others.25,26 Improved detection for platinum(II) has also gained attention.27−29 However, only few aptamer-based methods have been reported so far, which calls for the development of such a simple and selective platinum sensor for practical applications. We herein report an innovative aptameric system to improve the simplicity and selectivity of platinum detection based on the coordination of Pt to DNA base pairs. This novel technique encompasses a designed streptavidin aptamer sequence labeled with fluorescein isothiocyanate (FITC), in which nucleotides in the bulge and loop region are conservative, and several base pairs in the stem region are replaced with G-G mismatches in order to obtain a platinum controlled streptavidin-binding aptamer. The introduction of platinum sample is expected to lead to activation of the streptavidin (SA) aptamer sequence, allowing the binding to SA-coated magnetic beads/particles (MPs) for separation. The FITC label would subsequently react with anti-FITC-HRP (HRP, horseradish peroxidase), which can be monitored by a simple and direct reaction of enhanced CL HRP substrate (luminal reagent and peroxide solution) catalyzed by HRP (Figure S1). Using this system, other heavy-metal ions, such as Ca2+, Ni2+, Zn2+, Mn2+, Fe3+,

latinum-based chemotherapeutic regimens such as cisplatin, oxaliplatin, and carboplatin have been widely used for the treatment of tumors over the past 3 decades.1,2 The antineoplastic activity of platinum drugs is based on their interaction with cellular DNA leading to the formation of various types of adducts. In such systems, platinum likely attacks the drug’s primary target, DNA, and forms Pt-DNA adducts.3−6 Coordination occurs largely at the N7 positions of guanine (G) and adenine (A), with adjacent G residues being the most common binding sites. These Pt−base pairs can take the place of hydrogen-bonded Watson−Crick base pairs and enhance thermal duplex stability, while blocking DNA replication, transcription, and cell division. This has led to extensive investigation into Pt−base pairs for biomedical applications and sensor development.7−10 To gain a better understanding of the working mechanisms of platinum compounds, along with their metabolism and pharmacokinetics, new platinum detection strategies for use in the serum or other biological samples are under investigation. Such platinum assays should be feasible for a wide range of samples and should exhibit high selectivity for platinum ions against a background of competing analytes. To date, a series of analytical methods have been developed for measuring platinum in aqueous biological media, including spectrophotometry,11,12 atomic absorption spectrometry,13 inductively coupled plasma mass spectrometry (ICPMS),14,15 and HPLC/CE coupled to a range of detectors.16−18 However, the application of these methods for rapid on-site detection and in resource-limited settings was limited due to time-consuming methods, high costs, and large instruments. Recently, emerging technologies, such as those that use aptamers, have been widely © XXXX American Chemical Society

Received: July 25, 2015 Accepted: September 9, 2015

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DOI: 10.1021/acs.analchem.5b02810 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry Pb2+, Cd2+, Hg2+, Cu2+, and Ba2+, are not expected to show any notable effects on duplex stability. Thus, the development of a novel and highly selective platinum sensor that relies on the selective binding of Pt-base pairs could be envisaged. Moreover, the approach presented herein is expected to feature remarkable generality for other targets, including metal ions and proteins.

PBS, and MTT solution (5 mg/mL) was added to each well at 50 μg/well for 4 h and the produced formazan was solubilized for 15 min with DMSO. Absorbance was measured at 570 nm with a microplate reader. Cells treated with equivalent amounts of DMSO were used as controls. Assay of Cisplatin in Urine Samples. Eight-week old SD rats were obtained from Zhejiang Medical Science Academy (Hangzhou, China). The rats were provided with a standard pelleted food and water and were placed in metabolism cages. Rats treated with 10 mg/kg cisplatin by tail intravenous injection and treated with normal saline as a control. The urine was collected for 24 h and stored frozen until analyzed. Before measurement, the urine samples was dissolved in 70% nitric acid and 30% H2O2 for 1 h at 80 °C, then aqueous ammonia was added after cooling. The samples were diluted with water to a final concentration of 1% acid and centrifuged and filtered by a 0.22 μm filter.

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EXPERIMENTAL SECTION Apparatus. CL measurement was carried out using a PCcontrolled TECAN Infinite F 200 PRO. Absorbance was determined by an Amersham GenQuant Pro spectrophotometer. The photomicrographs were taken by Nikon’s A1 multiphoton confocal microscope. Reagents. All chemicals were of analytical grade and were used as received. The water was prepared using ELGA purelab option equipment. The streptavidin-coated magnetic particles (SA-MP, 1 μm, 5 mg/mL) were purchased from Polysciences, Inc. Potassium tetrachloroplatinate (II) (K2PtCl4) and anitFITC-HRP were bought from Sigma-Aldrich. HRP substrate kits were purchased from Millipore Corporation. Cisplatin and oxaliplatin were bought from from Kunming Guiyan Pharmaceutical Co. (Kunming, Yunnan, China). The 786-O renal cells were obtained from the Cell Culture Center (CCC, Beijing, China). Other chemical reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. Oligonucleotides were acquired from Invitrogen Biotechnology Co., Ltd. (Shanghai, China) and had the following sequences (Table 1).

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RESULTS AND DISCUSSION A schematic representation of CL detection of platinum(II) ions based on the coordination of Pt-base pairs to the aptameric system is shown in Scheme 1. The sensor consists of a designed Scheme 1. Schematic Representation of CL Detection of the Pt2+ Ion by Pt−Base Pair Coordination to the Aptamer

Table 1. DNA Sequences Used in This Work name aptamer A aptamer B aptamer C aptamer D aptamer E aptamer original

sequence 5′ -ATAGACCGCTGTGTGACGCAAGACTGTATFITC-3′ 5′ -ATAGACCGCTGTGTGACGCAAAACTATATFITC-3′ 5′ -ATAGACCGCTGTGTGACGCAAGACTATATFITC-3′ 5′ -ATAGACCGCTGTGTGACGCAAGAGTCTATFITC-3′ 5′ -AGAGACCGCTGTGTGACGCAACACTGTGTFITC-3′ 5′ -ATAGACCGCTGTGTGACGCAACACTCTATFITC-3′

SA aptamer sequence functionalized with a FITC fluorophore at the 3′- terminus. Two bases of the aptamer sequence were replaced with the G-G mismatch. In the presence of Pt2+ ions, platinum-mediated base pairs are formed between guanine residues in the aptamer sequence and activate its binding to the SA-coated beads. The FITC label is then attached to the surface, reacted with anti-FITC-HRP, and detected using an enhanced CL HRP substrate. In the absence of Pt2+ ions, the streptavidin aptamer remains in its inactive conformation, leading to a blank signal. The fluorescence and bright field photomicrographs (Figure 1) confirm that the designed aptamer was activated by the Pt2+ ions. Following incubation with Pt2+ ions, the SA-coated beads were observed in both the fluorescence and bright field photomicrographs (parts c and d of Figure 1, respectively), confirming binding of the FITC labeled aptamer probe onto the beads. In the absence of Pt2+ ions, no beads were observed in the fluorescence field (Figure 1a) due to aptamer blocking. The formation of adducts in DNA is the major cause of cancer cell death triggered by platinum-based anticancer drugs. To ensure that the platinum present in compounds such as K2PtCl4, cisplatin, and oxaliplatin has the same effect, cytotoxicity tests using MTT assays were carried out. Following treatment for 48 h with the desired platinum compounds (200

Assay Procedures on SA-MPs. In a typical experiment, 60 pmol of aptamer probes were mixed together with different amounts of Pt2+ or nontarget ions in 100 μL of PBS (8 mM Na2HPO4, 2 mM NaH2PO4, pH 7.4, 0.9% NaCl) for 0.5 h at 37 °C, then 2.5 μL of SA-MP was added and the mixture was incubated for 0.5 h at 37 °C. Second, the resultant SA-MPDNA conjugates were washed three times with 150 μL of wash buffer (8 mM Na2HPO4, 2 mM NaH2PO4, pH 7.4, 0.9% NaCl, 0.05% Tween 20), and 100 μL of 2 μg/mL anti-FITC-HRP were added and the mixture was incubated for 1 h at 37 °C. Finally, the resultant magnetic beads-DNA-HRP conjugates were then washed three times with 150 μL of wash buffer, and CL signals on the surface of the magnetic beads were detected directly with 100 μL of commercial CL HRP substrate. Cytotoxicity Test. 786-O renal cells were plated in quadruplicate at a density of 400 cells/well in 96-well plates and were allowed to adhere overnight. The cells were then treated with cisplatin, oxaliplatin, K2PtCl4, and FeCl3 at a concentration of 200 μM. After 48 h, cells were washed with B

DOI: 10.1021/acs.analchem.5b02810 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 1. Fluorescence (a, c) and bright field (b, d) photomicrographs of SA-coated beads before (a, b) and after (c, d) reaction with 100 μM Pt2+ ions and aptamer probes. Experimental conditions: aptamer probe was 60 pmol, SA-MPs was 200 ng.

μM) or iron ions (for the negative control group), the cell growth inhibition rates for 786-O renal cells were 63.65 ± 3.53, 81.92 ± 1.02, 70.49 ± 2.10, and 24.51 ± 2.28%, respectively (Figure 2). This indicates that platinum from K2PtCl4, cisplatin,

Figure 3. CL intensity (red) and blank signal (blue) vs different aptamer probes with G-G and G-A mismatches. Experimental conditions: Pt2+ ions were 6 μM, aptamer probe was 60 pmol, and SA-MPs and anti-FITC-HRP were 12.5 μg and 200 ng. The detection procedure was performed as described in the Experimental Section. Figure 2. Cytotoxicity test with different compounds. Experimental conditions: Pt2+ ions (K2PtCl4), cisplatin, oxaliplatin, and Fe3+ ion were 200 μM. The detection procedure was performed as described in the Experimental Section.

were conserved in the bulge and loop regions of the aptamer, we choose base pairs in the stem region as mismatch pairs for the Pt2+-DNA sequences. A comparison between aptamer probes bearing GG mismatched pairs in a number of stems was therefore carried out in parallel. Figure 4 shows that the CL intensity and CL ratio of aptamer probe A, bearing a single GG pair in each stem, were significantly higher than aptamer probe B, having two GG pairs in the stem between the bulge and loop regions, and aptamer probe C, having two GG pairs in the stem out of the bulge region. In aptamer probe A, the presence of

and oxaliplatin exhibits cytotoxicity due to DNA adduct formation. Inorganic platinum (K2PtCl4) has a lower inhibition rate than cisplatin and oxaliplatin, likely due to different cellular uptake rates.30,31 The Pt-DNA coordination occurs largely at the N7 positions of guanine (G) and adenine (A), with platinum compounds mainly partaking in intrastrand cross-links between the two adjacent guanine residues (GG), and between adjacent adenine and guanine residues (AG).7 Interstrand cross-links are also present but are less common. To evaluate the stability of the GPt-G and G-Pt-A adduct structures, which affects the binding ability of the designed aptamer sequences to streptavidin, the mismatch bases in the aptamer must be redesigned to improve the sensitivity and selectivity of the Pt2+ ions. Figure 3 shows a comparison between three designed aptamers bearing different mismatch pairs. Upon replacement of the GG pairs with GA pairs, the CL intensity decreased, which may indicate stronger Pt2+ intrastrand cross-links between the two adjacent GG residues than that between the AG residues. Moreover, no appreciable CL signal was observed in the control experiments using the original aptamer sequence as the aptamer probe. This suggests that the controlled streptavidin-binding aptamer was essential in signaling Pt2+ ions. We therefore selected an aptamer probe bearing two GG mismatches for subsequent experiments. The location of mismatched bases in the aptamer sequence is also critical for a successful Pt2+ assay system. As nucleotides

Figure 4. CL intensity (red) and CL ratio (blue) vs different aptamer probes with G-G in different location. Experimental conditions: Pt2+ ions were 6 μM, aptamer probe was 60 pmol, and SA-MPs and antiFITC-HRP were 12.5 μg and 200 ng. The detection procedure was performed as described in the Experimental Section. C

DOI: 10.1021/acs.analchem.5b02810 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry GG pairs on different stems may lead to a lower steric hindrance in the presence of Pt2+ and a more incomplete aptamer sequence structure in the absence of Pt2+, resulting in higher CL intensities and ratios. Aptamer probe A was therefore selected for the following studies. We then investigated the thermodynamics of this platinumoligonucleotide coordination aptasensor by monitoring the CL intensity and changes in the ratio with variation in temperature, as shown in Figure S2. The detection probe, i.e., the platinumcontrolled SA aptamer, exhibited higher stability at lower temperatures, resulting in stronger CL intensities. However, the affinity of the aptamer sequence to SA-coated MPs and antiFITC-HRP to the FITC label require higher reaction temperatures. At temperatures below 37 °C, the SA aptamer is relatively stable (melting point ∼40 °C), thus the CL intensity and ratio gradually increased with increasing temperature. However, above 37 °C, thermal destabilization of the SA aptamer duplex structure occurred, resulting in low CL emission. Optimization of Reaction Conditions. Several parameters were investigated to establish optimal conditions for CL detection of the Pt2+ ions, including the amounts of SA-MPs, aptamer probe, anti-FITC-HRP, and Mg2+ used in the system. As shown in Figure S3, an increase in SA-MP gave an increase in CL intensity up to 12.5 μg of SA-MP, after which the CL intensity decreased, likely due to quenching by an inner filter effect of excess SA-MP. Subsequent studies therefore employed 12.5 μg of SA-MP. The effects of different amounts of aptamer probe on CL intensity are shown in Figure S4. CL intensity increased between 10 and 60 pmol of aptamer probe, then decreased gradually, likely due to progressive saturation of the SA-MP binding sites and the excess of aptamer probe with incomplete aptamer sequence giving a higher blank. Thus, 60 pmol was selected as the optimal aptamer probe quantity for subsequent experiments. CL intensity increased between 20− 200 ng of anti-FITC-HRP, after which point it reached a plateau (Figure S5), as the noise increased rapidly in comparison to the signal. Thus, 200 ng of anti-FITC-HRP was used in subsequent studies. It is generally recognized that the addition of the reaction buffer MgCl2 can have a significant impact on the binding properties of aptamers. Its effect was monitored by varying the amount of Mg2+ ions used in the process (Figure S6), with CL intensity being higher in the presence of 2.5−10 mM MgCl2. Above this concentration, CL intensity dropped once again. Selectivity. The selectivity of the sensor toward Pt2+ ions was investigated by recording changes in CL intensity with the addition of Ca2+, Ni2+, Zn2+, Mn2+, Fe3+, Pb2+, Cd2+, Hg2+, Cu2+, and Ba2+. As indicated in Figure 5 (solid colored boxes), the sensor showed a negligible response to other metal ions at 100 μM when compared to its response to Pt2+ at 10 μM. In addition, 10 μM of Pt2+ and 100 μM of each metal ion were added together in PBS. The CL signal response of the Pt−M metal ion pair (Figure 5, gray bars) suggests excellent selectivity for Pt2+ over other metal ions, owing to the high affinity and specificity of Pt−base pairs. These results indicate that our proposed biosensor appears to meet the selective requirements for biomedical and environmental applications. Quantification of Pt2+ Ions. The quantitative behavior of this assay under the optimized experimental conditions was assessed by monitoring the dependence of the CL intensity on the amount of Pt2+ ions. As shown in Figure 6, CL intensity was proportional to the concentration of Pt2+ ions. The linear curve

Figure 5. Selectivity of the analysis of Pt ions by the method depicted in Scheme 1 with the following metal ions: 1, Pt2+; 2, Ca2+; 3, Ni2+; 4, Zn2+; 5, Mn2+; 6, Fe3+; 7, Pb2+; 8, Cd2+; 9, Hg2+ ; 10, Cu2+; and 11, Ba2+. The concentration of Pt2+ was 24 μM (10 μg/mL). The concentrations of the other metal ions were 100 μg/mL. Other experimental conditions were the same as Figure 6.

Figure 6. Log−Log calibration data for Pt2+ ions. Experimental conditions: aptamer probe 60 pmol, SA-MPs and anti-FITC-HRP 12.5 μg and 200 ng, respectively. The detection procedure was performed as described in the Experimental Section.

fitted a regression equation of Lg I = 0.5677 Lg C + 3.6782 within a range from 0.12 to 120 μM with a correlation coefficient of R = 0.9949, where I is the CL intensity and C is the concentration of Pt2+ ions. The limit of detection was 60 nM at a signal-to-noise ratio of 3, with a large dynamic range that spanned approximately 4 orders of magnitude. The sensitivity of this assay compares favorably with previous efforts for platinum detection (Table S2). For example, a previously reported fluorescence method offered a detection limit of 0.72 μM,11 while an electrochemical approach yielded a 0.5 μM limit of detection (LOD).32 Use of atomic absorption spectrometry allowed Brouwers to establish a method with an LOD of 0.1 μM.13 Furthermore, low nM (ng/mL) detection limits were generally obtained using HPLC coupled with inductively coupled plasma mass spectrometry.15−17 In addition, Hernandez-Santos et al. investigated the deposition of silver on a Pt-modified (from cisplatin) electrode and found that the analytical signal corresponded to the anodic stripping of the deposited silver, achieving cisplatin detection at 0.5 μM.33 However, the assay reported here is exceptionally rapid (