Label-Free Fluorescent Copper Nanoclusters for Genotyping of

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Label-free fluorescent copper nanoclusters for genotyping of deletion and duplication of Duchenne muscular dystrophy Chung-An Chen, Chun-Chi Wang, Yuh-Jyh Jong, and Shou-Mei Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00918 • Publication Date (Web): 18 May 2015 Downloaded from http://pubs.acs.org on May 21, 2015

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Analytical Chemistry

Label-free fluorescent copper nanoclusters for genotyping of deletion and duplication of Duchenne muscular dystrophy Chung-An Chena, Chun-Chi Wanga, Yuh-Jyh Jongb,c,d, Shou-Mei Wu*,a,e a

School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan c Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan d College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan e Department of Chemistry, College of Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan b

ABSTRACT: Real applications in clinical diagnosis of label-free fluorescent copper nanoclusters (CuNCs) are demonstrated. Double-strand DNAs (dsDNA) can act as effective templates for the formation of CuNCs, which can be used to distinguish the deletion or duplication genotypes of Duchenne muscular dystrophy (DMD) due to different fluorescent intensities. After PCR, the DMD amplicons reacted with copper ion by reduction of ascorbic acid and generated fluorescence. The exons of DMD gene were taken as the model analytes for genetic diagnosis. In this sensing system, the deletion type does not show fluorescence; on the other hand, the duplication type emits higher fluorescence than normal type. Parameters of this sensing system were optimized, including PCR conditions, levels of copper ion and ascorbate and reaction time. The DMD-dominated exons 45, 46 and 47 were detected, and applied to 6 samples of DMD patients. The results were consistent with the MLPA method. This strategy was feasible to detect all exons of this disease.

INTRODUCTION Fluorescent-labeling usually increases the background interference of primers. Recently some fluorescent nanomaterials, such as copper nanoparticles/ copper nanoclusters (CuNPs/ CuNCs),1-4 silver nanoclusters,5-7 gold nanoclusters,8,9 carbon dots,10,11 and polymer dots,12-14 have drawn interest in biosensing and bioimaging due to unique optical, electronic and catalytic properties. The dsDNA template can support the formation of CuNPs at low concentration of CuSO4 and the formation of CuNPs results in high fluorescence.1 The CuNPs exhibits fascinating features, including ease of synthesis, high water solubility, low toxicity, biocompatibility and excellent stability.2 It would be interesting to explore the design and construct a biosensing system using dsDNA-CuNPs as fluorescence probes for DNA analysis.3 Duchenne muscular dystrophy (DMD) is a common Xchromosomal recessive disorder caused by mutations in the dystrophin gene.15,16 DMD affects 1 in 3,500 to 1 in 6,000 male births.17-19 This gene is located on Xp.21 spanning 2.4 Mb of genomic DNA, containing 79 exons that encode a 14kb mRNA and is the largest gene in humans.20,21 According to extant literature, deletion of one or more exons occurs in about 60~65% of cases of DMD and duplication(s) of one or more exons was observed in about 5~10% DMD cases. 22,23 The remaining DMD cases, accounting for approximately 30~35% of cases, were caused by single point mutations, small deletions or insertions.23,24 Previous research on the breakpoints of deletion indicates that intron 44 is the frequent starting breakpoint.25 Therefore, exon 45 is the most commonly deleted ex-

on in DMD patients and large deletions of exons 45-47 often happen.25 Mutation-screening strategies for analysis of DMD gene tend to concentrate on monitoring of large deletions or duplications by Southern blotting,21,22 multiplex polymerase chain reaction (multiplex PCR),26,27 quantitative PCR,28,29 multiplex amplifiable probe hybridization (MAPH),30 multiplex ligationdependent probe amplification (MLPA),31-33 capillary gel electrophoresis (CGE),34,35 or gold nanoparticles (AuNPs).36 DNA sequencing is another technique for identification of point mutations but it is still laborious and time-consuming. However, some of them require PCR product purification or hybridization steps which makes them more complex. Here we tried to establish a new method for diagnosis of deletion and duplication types of DMD. It is the first method that proposes use of fluorescent CuNCs for genotyping. Exon 46 of DMD is tried in this sensing system and is then extended to other exons.

EXPERIMENTAL Chemicals and materials. Oligonucleotide sequences are listed in Table S1. All oligonucleotides were designed ourselves, ordered from MD Bio, Inc (Taipei, Taiwan) and stored as 100-μM stock solutions in sterilized water at -20℃. 3Morpholinopropane-1-sulfonic acid (MOPS), 3-(NMorpholino) propanesulfonic acid sodium salt (MOPS sodium salt) and CuSO4 were obtained from SigmaAldrich (Sigma, St. Louis, MO, USA). Sodium chloride (NaCl), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were from E. Merck

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(Darmstadt, Germany). The double-distilled water was obtained from a Milli-Q water system (Millipore, Milford, MA, USA). Assay procedures of PCR and sensor preparation. The total PCR volume was 50 μL containing a mixture of 50 ng of genomic DNA, 2.5 mMd NTP (TaKaRa, Shiga, Japan), 1× PCR buffer (TaKaRa), 160 nM of each primer and 2.0 U of e2TAK DNA polymerase (TaKaRa). The PCR amplification was performed in a Thermocycler (Biometra, Göttingen, Germany) with an initial denaturation at 95℃ for 10 min, followed by 40 cycles of denaturing at 95℃ for 30 sec, annealing at 59℃ for 15 sec, extensing at 72℃ for 30 sec, and a final extension at 72℃ for 10 min. After PCR, the product was mixed with 10 mM MOPS buffer, 150 mMNaCl, 4 mM sodium ascorbate and 500 μM CuSO4. The final volume was 200 μL, which was reacted for 3 min. at room temperature. Apparatus. The measurements were carried out on an F4500 fluorescence spectrometer (Hitachi, Japan) with excitation and emission slit set at 5 nm and 20 nm, respectively. Scanning speed was set on 240 nm/ min. A quartz cell with an optical path length of 1.0 cm was used. All measurements were at room temperature, unless stated otherwise. The fluorescence emission of the system was recorded from 500 to 650 nm at the excitation wavelength of 345 nm. Transmission electron microscopy (TEM) measurements were performed on a JEOL JEM-1400 transmission electron microscope (JEOL, Japan) at 100 kV. TEM samples were prepared on carbon-

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coated copper grid substrates and were baked in an oven at room temperature. Real sample application. DNA samples were collected from peripheral whole blood of DMD patients and normal individuals in Kaohsiung Medical University Hospital. The ethical approval of this study was obtained from the Institutional Review Board at Kaohsiung Medical University Hospital, where participants were recruited. Written informed consents were obtained from all participants.

RESULTS AND DISCUSSION Design and principle of the biosensor for the exon of DMD detection. A special sensing sequence was designed at forward and reversed primers (Fig. 1A). After PCR, the designed duplex DNA reacted with copper ions under the reduction of sodium ascorbate. The dsDNA-CuNCs complex emits fluorescence. The deletion type could not form amplicones and showed no fluorescence. In the duplication type, fluorescence is higher than the normal type. The dsDNA-CuNCs can be formed with maximal excitation and emission at 345 and 565 nm, respectively (Figure 1B).1,2 The formation of dsDNA and CuNCs produces fluorescence (Fig. 1B). Without dsDNA, fluorescent signal is not produced, even if the primers exist at PCR reaction for copper ion and sodium ascorbate in the buffer. On the other hand, we also confirmed that the background could not produce any interference in this reaction (Figure 1C).

Figure 1. (A) Schematic representation of the sensing procedure for genotyping of dsDNA-CuNCs. (B) Fluorescence excitation (blue line) and emission (red line) spectra of the obtained dsDNA-CuNCs.; (C) The fluorescence spectra of the sensing system under different conditions: (a) Normal exon 46 (after PCR); (b) blank (PCR without genome); (c) exon 46 forward

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primer; (d) exon 46 reversed primer; (e) 10 mM MOPS + 150 mMNaCl; (f) 10 mM MOPS + 150 mMNaCl (without copper ion and sodium ascorbate); Cu2+ concentration: 500 μM; sodium ascorbate concentration: 4 mM.

Figure 2. TEM images of dsDNA-CuNCs with different DNA samples: (A) no DNA, (B) normal type, (C) deletion type, (D) duplication type.

Figure 3. Effects of concentration of primers and number of cycles of PCR reaction on the fluorescence of the dsDNA-

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CuNCs in the PCR reaction. (A) 120 nM of forward and reversed primers; (B) 160 nM of forward and reversed primers; (C) 200 nM of forward and reversed primers; (D) 30 cycles; (E) 35 cycles; (F) 40 cycles. DNA types: (a) Duplication type; (b) Normal type; (c) Deletion type; (d) Blank. Other conditions were the same as in Figure 1. The CuNCs were characterized with transmission electron microscope (TEM) (Figure 2) which supports the nanoparticles formation with low concentration of CuSO4. It was observed that the size of dsDNA-CuNCs was less than 10 nm. Other TEM images are provided as supporting information (Figure S-1). The results show that the sintering of CuNCs was significant (Figure 2). Figure 2A shows the particles without DNA sample mixed with copper ion and sodium ascorbate and the CuNCs were produced rarely. For DMD deletion sample, some of the CuNCs were observed in the sintering action, but it was not obvious (Figure 2C). Figure 2B and 2D show normal and duplication type DNA samples. After amplification by PCR reaction and mixing with copper ion and sodium ascorbate, the increase of particle size of CuNCs (Figure 2B) and a seriously sintering action in duplication type sample (Figure 2D) were clearly observed. Based on the above data, we considered that the fluorescence is related to the amounts of CuNCs formed. Therefore, it can be proved that the different fluorescent intensities are present in real DNA samples with normal, deletion and duplication type (Figure 2). Optimization of sensing system. Primers and PCR cycles were examined and the results (Figure 3) show that more or less primers were not proper for PCR amplification. Therefore, 160 nM of primers were used (Figure 3B). Besides, the number of PCR cycles influenced the yield of PCR products. We tested 30, 35 and 40 cycles (Figures 3D-F). The results show that the fluorescent intensity in 30 cycles was very low, indicating the insufficient amount of dsDNA. However, in 40 cycles, the blank showed some signals of fluorescence. Thirty five cycles were selected as the best optimal condition. The effect of Cu2+ ion was found to be an important factor affecting fluorescent sensitivity for formation of dsDNACuNCs. Morkhir et al. reported that dsDNA-CuNCs might be formed in these steps: the reaction of reducing copper (II) to copper (I) followed by the disproportionation of copper (I) into copper (II) and copper (0), and clustering of the latter on dsDNA producing stable nanoparticles.1 Figure S-2A shows that weak a fluorescent signal was acquired when using 250 μM Cu2+ and no significant differences were observed between the use of 500 μM and 1000 μM Cu 2+ ion. However, copper (II) complexes have been reported previously to cleave DNA via oxygen-based radicals.37 With the concentration of Cu2+ higher than 500 μM, mixture of Cu2+ and sodium ascorbate might generate hydroxyl radicals causing destruction of double DNA helix.1 The concentration of 500 μM Cu2+ was chosen in all further experiments. The concentration of sodium ascorbate had to be investigated. As shown in Figure S-3, the highest fluorescent signal was achieved at 4 mM of sodium ascorbate. When it was higher than 4 mM, the fluorescent intensity decreased. Therefore, 4 mM sodium ascorbate was used in this study. The incubation time was also investigated (Figure S-4). At 3 min, the fluorescent intensity reached the maximum, then decreased afterwards. It is also observed that the duplication type had about 600 RFU of fluorescent intensity which was

higher than normal type. These results can be used to diagnose the deletion or duplication type of DMD. Application of other exons and the diagnosis of real samples. After using exon 46 as the model target, we also tested exons 45 and 47 by this label-free fluorescent CuNCs (Figure 4). The data indicate it is feasible to detect deletion and duplication types with this strategy. All the 6 clinical samples acquired from hospitals had the same results as the MLPA method. The outcomes contained 5 deletion types and 1 duplication type from exons 45 to 47, respectively (Table 1). This result indicates that the method can be useful for DMD diagnosis. Furthermore, we expect this method to be applied widely for detection of all exons in DMD diagnosis.

Figure 4. Effects of the dsDNA-CuNCs assay for different exons test. (A) Exon 45; (B) Exon 47. (a) Duplication type; (b) Normal type; (c) Deletion type; (d) Blank. Other conditions were same as Figure 1. Table 1. Discrepant results of gene type of DMD by MLPA and dsDNA-CuNCs.

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Subjects (Patients No.)

MLPA

dsDNA-CuNCs

1

del exon 45-47

del exon 45-47

2

del exon 45-47

del exon 45-47

3

del exon 45-47

del exon 45-47

4

del exon 45-47

del exon 45-47

5

del exon 45-47

del exon 45-47

6

dup exon 45-47

dup exon 45-47

*del

= deletion; dup = duplication

CONCLUSIONS It is the first dsDNA-CuNCs designed for application in PCR reaction and analysis of a real clinical sample. Using dsDNA-CuNCs as fluorescent probes, we successfully establish a rapid, simple and cost-effective method for the exons of DMD diagnosis. This method can display sensitive and selective assays to distinguish the genotype of deletion or duplication for DNA of DMD patients. Besides, the process avoids the need of designing complicated fluorescent primers and using the DNA clean-up kits to purify the PCR products. This study provides a new detection method which has the potential to be a general method for genotyping. It also provides a new opportunity for PCR design for fluorescent bioanalytical assay for using dsDNA-CuNCs as a signal transition sensor.

ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Tel: (+886) 07-3121101 ext 2164; Fax: (+886) 07-315-3159597; E-mail: [email protected]

ACKNOWLEDGMENTS We deeply extend our sincere thanks to the volunteers who kindly contributed samples that were crucial to this study. We gratefully acknowledge the support of the Ministry of Science and Technology of Taiwan (MoST), Kaohsiung Medical University, Instrument Center of National Cheng Kung University and NSYSUKMU 104-I006 joint research project (#NSYSUKMU104-I006) by way of funding of this work and the help of Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.

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Label-Free Fluorescent Copper Nanoclusters for Genotyping of

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