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Multiplex DNA sensor for BRAF and BRCA detection
Analytical Biochemistry 438 (2013) 22–28
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Multiplex DNA sensor for BRAF and BRCA detection Xiangzhao Ai, Qiang Ma, Xingguang Su ⇑ Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 20 November 2012 Received in revised form 27 February 2013 Accepted 28 February 2013 Available online 13 March 2013 Keywords: Multiplex DNA sensor Quantum dots BRAF DNA BRCA DNA Simultaneous detection
a b s t r a c t In this article, a kind of simple, sensitive, and rapid quantum dots (QDs)-based multiplex DNA sensor is developed for the simultaneous detection of BRAF and BRCA DNA based on the ‘‘nano-on-micro’’ technique. In our strategy, capture DNABRCA and DNABRAF are simultaneously immobilized on the surface of amino-modified silica microbeads. After blocking with bovine serum albumin (BSA), different concentrations of target DNABRCA and DNABRAF are introduced to hybrid with complementary capture DNABRCA and DNABRAF. After hybridization, QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA were added into the above solution so that the unreacted capture DNABRCA and DNABRAF could be detected by QDs657-labeled probe DNABRCA and QDs546-labeled probe DNABRAF simultaneously. We demonstrate that the proposed method is effective for detecting BRAF and BRCA DNA with high sensitivity. The sensor has great potential to expand its application to the early diagnosis of cancers such as breast cancer, ovarian cancer, and papillary thyroid carcinoma. Ó 2013 Elsevier Inc. All rights reserved.
Recently, development of life science requires more sensitive and rapid analysis methods for various biomolecules, including protein, DNA, virus, and cells [1–4]. In these novel methods, optical techniques have been efficiently employed due to their fast, cheap, and high-throughput properties. Most optical analytical systems are based on various dyes, for example, cyanine-type and Alexa Fluor-type fluorophores [5]. Unfortunately, these organic dyes suffer from inherent disadvantages such as photobleaching, broad excitation and emission bands, small Stokes shift, and poor environmental stability. These properties significantly affect the uniformity of fluorescent signals and have some limitations for the trace analysis of biomolecules. Semiconductor quantum dots (QDs)1 are under intense attention based on their predominant optical properties. First, they have high quantum efficiency and a continuous absorption profile [6,7]. This allows different QDs emissions to be excited simultaneously using a single wavelength. Second, they have size-tunable emission spectra that make different-colored QDs (from blue to red) obtained by simple refluxing in different times [8]. Third, the resistance to photobleaching and high stability of QDs in the biological environment [9,10] permit QDs to be used in monitoring biological molecules in vivo. The advantages of QDs, ⇑ Corresponding author. E-mail address: [email protected] (X. Su). Abbreviations used: QDs, quantum dots; PTC, papillary thyroid carcinoma; AMSM, amino-modified silica microbead; BSA, bovine serum albumin; MPA, 3-mercaptopropyl acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; sulfo-NHS, N-hydroxysulfosuccinimide; TEOS, tetraethoxysilane; APTES, 3-aminopropyltrimethoxysilane; THPMP, 3-(trihydroxysilyl)-propylmethylphosphonate (THPMP); WB, washing buffer; HB, hybridizing buffer; PBS, phosphate buffer solution; PL, photoluminescence; RSD, relative standard deviation. 1
0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.02.029
such as narrow, symmetric, tunable emission spectra and large Stokes shift, enable them to be an excellent optical material for multiplex analysis. For instance, Freeman and coworkers reported on the use of T-rich aptamer and T-rich aptamer-modified QDs for the selective analysis of Hg2+ or Ag2+ ions using an electron transferquenching path [11]. Regarding the need for sensitive and multiple DNA detection in gene therapy and disease forecasting, many efforts have focused on the development of different DNA biosensors capable of measuring multiple targets in parallel. There are two broad classes of techniques used for multiplexing assay: planar arrays [12] and suspension arrays [13]. Different from conventional planar arrays, the advantages of suspension arrays include (i) enhanced reaction kinetics due to their suspension in homogeneous solution; (ii) the ability to measure analyte response to several different targets simultaneously; (iii) shorter incubation and assay time, leading to higher throughput; and (iv) higher sensitivity and lower cost. Thus, suspension arrays are suitable for multiplex assays and signal amplification for trace DNA detection. For instance, Jang and coworkers designed a high-sensitive multiplexed DNA detection assay using oligonucleotide-modified magnetic microparticle and gold nanoparticle probes that can simultaneously detect three kinds of DNA sequences in one sample [14]. Luo and coworkers developed a sensitive, magnetic, and multiplexed DNA detection biosensor for simultaneous detection of two target DNA sequences [15]. Owing to its unique properties, multiplexed DNA detection assay has shown great promise in clinical diagnostics. In this article, a novel, sensitive, and multiplexed DNA biosensor is proposed based on the ‘‘nano-on-micro’’ principle, which means
DNA sensor for BRAF and BRCA detection / X. Ai et al. / Anal. Biochem. 438 (2013) 22–28
that nanoparticles are conjugated on the surface of microbeads [16]. Compared with other methods, the nano-on-micro technique has two advantages in multiplex analysis and immunoassay for biomolecules. First, the large surface areas of microbeads make it possible to capture 100,000 analytes on 5.5-lm microbeads, which prominently improves the sensitivity for analysis [17]. In addition, analyte can be captured in a short time based on enhanced reaction kinetics of microbeads [18]. Here, we combine QDs with silica microbeads based on the nano-on-micro principle to develop a multicolor probe for simultaneous detection of two kinds of DNA: BRCA and BRAF. BRCA is a susceptive mutation DNA in human breast cancer that is responsible for most hereditary breast and ovarian cancers [19,20]. Moreover, it has been reported that missense BRAF mutation occurs in 29% to 83% of papillary thyroid carcinoma (PTC), which is the most common malignant tumor of the thyroid gland and the most frequent type of endocrine cancer [21]. Therefore, the detection of special base sequences in human genome such as BRCA and BRAF DNA is a crucial research topic for the effective treatment of malignant tumors, including breast cancer, ovarian cancer, and PTC, in the medical field. A few methods have been reported for the detection of BRCA and BRAF DNA. However, these methods have some inherent limitations. For instance, Li and coworkers reported a novel sandwich-type assay for the optical detection of BRCA DNA [22], but it has only a single signal emission that is hardly expanded to multiplexed DNA detection assay. Liao and coworkers presented a sensitive technique for the electrochemical detection of BRAF DNA [23], but it has an intricate mechanism and a convoluted electrochemical analysis procedure. Here, we propose a novel simultaneous detection strategy for BRCA and BRAF DNA based on the nano-on-micro technique, as shown in Scheme 1. First of all, capture DNABRCA (blue) and DNABRAF (purple) are simultaneously immobilized on the surface
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of amino-modified silica microbeads (AMSMs) by glutaraldehyde. After blocking with bovine serum albumin (BSA), different concentrations of target DNABRCA (red) and target DNABRAF (dark green) are introduced to hybrid with complementary capture DNABRCA and DNABRAF for 10 h. After hybridization, QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA were added into the above solution so that the unreacted capture DNABRCA and DNABRAF could be detected by QDs657-labeled probe DNABRCA (red light emission) and QDs546-labeled probe DNABRAF (green light emission) simultaneously. To the best of our knowledge, this is the first report of the detection of BRCA and BRAF DNA simultaneously with the nano-on-micro principle. This novel sensor has great potential to expand its application to the early diagnosis of cancers such as breast cancer, ovarian cancer, and PTC. Materials and methods Materials and reagents All chemicals used were of analytical reagent grade without further purification. 3-Mercaptopropyl acid (MPA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) were purchased from J&K Chemical. Tellurium powder, CdCl2, NaBH4, Triton X-100, cyclohexane, n-hexanol, tetraethoxysilane (TEOS), 3-aminopropyltrimethoxysilane (APTES), 3-(trihydroxysilyl)-propylmethylphosphonate (THPMP), isopropyl alcohol, NaCl, and BSA were purchased from Sigma–Aldrich. The water used in all experiments had a resistivity higher than 18 MX cm. The oligonucleotides were purchased from Dingguo Biotechnology (Beijing, China) and purified using high-performance liquid chromatography. The sequences of oligonucleotides are shown in Table 1. In addition, washing buffer (WB, 10 mmol L1 Tris–HCl,
Scheme 1. Illustration of the mechanism of simultaneous detection of BRAF and BRCA DNA with dual-color QDs based on ‘‘nano-on-micro’’ technique.
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DNA sensor for BRAF and BRCA detection / X. Ai et al. / Anal. Biochem. 438 (2013) 22–28
pH 7.4), hybridizing buffer (HB, 10 mmol L1 Tris–HCl [pH 7.4] and 0.1 mol L1 NaCl), and phosphate buffer solution (PBS, 10 mmol L1, pH 7.4) were used in the whole following process. Instruments Fluorescence experiments were performed on an RF-5301 PC spectrofluorophotometer (Shimadzu, Japan), and a 1-cm pathlength quartz cuvette was used to measure the fluorescence spectrum. A bath ultrasonic cleaner (Autoscience AS 3120, Tianjin, China) was used to disperse the silica microbeads. All pH measurements were completed using an Ohaus STARTER 2100/3C meter (USA). An inverted fluorescence microscope (Olympus FV1000 IX71) equipped with a multispectral imaging system (Nuance, CRI, Woburn, MA, USA) was used to observe the fluorescence change of QDs. All optical measurements were carried out at room temperature under ambient conditions, and the excitation wavelength was 450 nm. Synthesis of AMSMs In the current study, AMSMs were prepared by the reverse microemulsion method at room temperature. In this method, cyclohexane was used as a continuous phase and Triton X-100 and n-hexanol were regarded as surfactant and cosurfactant, respectively. We mixed 15 ml of cyclohexane, 3.6 ml of Triton X100, 3.6 ml of n-hexanol, 800 ll of water, and 100 ll of TEOS to form a microemulsion system. After stirring for 30 min, 100 ll of NH4OH was added in solution to initiate the hydrolysis of TEOS. The reaction progressed in the dark with stirring for 24 h. Then, 30 ll of APTES and 80 ll of THPMP were injected into the reaction system, which was kept under stirring for 1 more day. Finally, the microemulsion was broken by adding 20 ml of acetone and the resultant precipitate was AMSMs, which were washed in sequence with ethanol and water several times. During each washing procedure, AMSM dispersion was first subjected to high-velocity centrifugation (10,000 rpm, 10 min), followed by decantation of the supernatant and redispersion of the precipitate in the next solvent with the aid of supersonication. Ultimately, aqueous AMSMs were dispersed in 1 ml of water and preserved in a refrigerator at 4 °C for further experiments. The mass concentration of AMSMs was 0.0548 g ml1. Preparation of CdTe QDs-labeled probe DNA CdTe QDs were synthesized by refluxing routes as described in detail in Ref. [24]. Briefly, the precursor solution of CdTe QDs was formed in water by adding fresh NaHTe solution to 1.25 103 mol L1 N2 saturated CdCl2 solution at pH 11.4 in the presence of MPA as stabilizing agent. The molar ratio of Cd2+/MPA/HTe was charged at 1:2.4:0.5. The CdTe precursor solution was subjected to reflux at 100 °C under open-air conditions with a condenser attached, and different sizes of CdTe QDs were obtained at different
refluxing times. The photoluminescence (PL) emission wavelengths of CdTe QDs used in this study were 546 and 657 nm. QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA were prepared by covalent link carboxyl and amino, respectively [25]. Briefly, 0.3 ml of 2.5 106 mol L1 EDC and 0.2 ml of 1.5 106 mol L1 sulfo-NHS were added into as-prepared 0.5 ml of QDs solution (Em 546 or 657 nm) to activate carboxyl of QDs with stirring for 0.5 h. Then, 100 ll of 1 106 mol L1 probe DNABRCA or probe DNABRAF was injected and stirred for 3 h to form a firm amide bond. Finally, 2 ml of isopropyl alcohol was added to precipitate QDs-labeled probe DNA, and unreacted reagents were removed by high-velocity centrifugation (10,000 rpm, 10 min). Ultimately, 1 ml of aqueous dispersed QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA was preserved in a refrigerator at 4 °C for further experiments. Preparation of capture DNA-modified silica microbeads In this study, capture DNABRCA and DNABRAF were linked with the amino groups on the surface of AMSMs by glutaraldehyde [26,27]. First, 40 ll of prepared AMSMs was suspended in 1 ml of PBS (2.2 mg ml1). Then, 100 ll of 1% glutaraldehyde was added to the solution, vibrating for 6 h. Remanent glutaraldehyde was removed by centrifugation, and the precipitation was redispersed in 1 ml of PBS by ultrasonic cleaner. Next, 25 ll of 1 106 mol L1 capture DNABRCA and 25 ll of 1 106 mol L1 capture DNABRAF were injected at room temperature and kept under stirring overnight. After that, 2 mg of NaBH4 was added, and the mixture was vibrated at room temperature for 2 h. The unreacted reagents were removed by centrifugation, and the precipitation was redispersed in 1 ml of PBS. Then, 1% BSA solution (10 mg ml1) was injected to block the unreacted sites on the surface of silica microbeads for 1 h, and remanent BSA was removed later. The precipitation was redispersed in 1 ml of HB solution by ultrasonic cleaner. Simultaneous detection of two target DNAs The immunoassay technique was used in this study to detect two target DNAs simultaneously. First, various concentrations of target DNABRCA and target DNABRAF were incubated with two capture DNA-modified AMSMs for 10 h at room temperature. Then, 400 ll of QDs546-labeled probe DNABRAF and 400 ll of QDs657-labeled probe DNABRCA were injected into the above solution and shaken overnight. Unreacted reagents were removed by centrifugation, and the precipitation was redispersed in 1 ml of HB solution after washing several times with WB solution. The PL intensity in this experiment was recorded with an excitation wavelength of 450 nm. For the real sample detection, the human serum samples were collected from healthy adult volunteers in a local hospital and deproteinized by centrifugation at 14,000 rpm for 10 min after adding acetonitrile in serum samples (CH3CN/serum, 1:1). Finally, all supernatant serum samples were subjected to a 10-fold dilution
Table 1 Sequences of oligonucleotides used in this study.
a
Name of oligonucleotide
Gene
Sequence of oligonucleotide (50 –30 )
Capture DNABRAF Probe DNABRAF Target DNABRAF One-base mismatched target DNABRCAa
BRAF BRAF BRAF BRAF
50 -GATTTTGGTCTAGCTACAGAGAAATCTCGA-(CH2)3-NH2-30 50 -TCGAGATTTCTCTGTAGCTAGACCAAAATC-(CH2)3-NH2-30 50 -TCGAGATTTCTCTGTAGCTAGACCAAAATC-30
Capture DNABRCA Probe DNABRCA Target DNABRCA Two-base mismatched target DNABRCAa
BRCA BRCA BRCA BRCA
50 -TCGAGATTTCACTGTAGCTAGACCAAAATC-30 50 -GAAACCCTATGTATGCTCTTTTTTTTTT-(CH2)3-NH2-30 50 -GAGCATACATAGGGTTTCTCTTGGTTTCTTTGATTATAATTCATAC-(CH2)6-NH2-30 50 -GAGCATACATAGGGTTTCTCTTGGTTTCTTTGATTATAATTCATAC-30 50 -GAGCATACATTGGGTTTCTCTTGGTTTCTTTGATTATTATTCATAC-30
The mutant nucleotide in the BRAF and BRCA mismatched target DNA is indicated in boldface and underlined.
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in 1 ml of HB solution, and different concentrations of target DNABRAF and DNABRCA were added into it to prepare the spiked samples. Results and discussion Fluorescence spectra of QDs and hybridization system Spectra overlap is a limiting factor in multiplex target detection using optical techniques. For the purpose of minimizing spectral interference, green CdTe QDs (Em 546 nm) and red CdTe QDs (Em 657 nm) were used in our experiment for labeling probe DNABRAF and probe DNABRCA, respectively. Fig. 1A shows the normalized emission spectra obtained from two kinds of CdTe QDs. It can be seen that the distance between the maximum emission peaks of two QDs is 111 nm, which is enough to detect two target DNAs without any spectral interference. Fig. 1B shows the normalized fluorescence spectra of bare AMSMs without DNA (curve a), bare QDs incubated with capture DNA-modified AMSMs (curve b), QDs-labeled probe DNA incubated with capture DNA-modified AMSMs in the absence of target DNA (curve c), and QDs-labeled probe DNA incubated with capture DNA-modified AMSMs in the presence of target DNA (curve d). It can be seen that the PL intensity of probe DNA-linked QDs (curves c and d) is much greater than that of bare QDs (curve b) after incubation with capture DNA modified on the surface of AMSMs, indicating that QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA can be successfully immobilized on the surface of AMSMs via DNA hybridization. In the absence of target DNA, as shown in curve c, all of the capture DNA on the surface of AMSMs could hybridize completely with complementary QDs-labeled probe DNA, so the PL intensity is the strongest. However, in the presence of target DNA, as shown in curve d, some capture DNA hybridizes with target DNA and only the unreacted capture DNA on the surface of AMSMs can hybridize with QDs-labeled probe DNA, so the PL intensity decreased. In addition, the weaker fluorescence intensity of bare QDs (curve b) after incubation with capture DNA modified on the surface of AMSMs is due to the effect of physical adsorption.
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strength on the PL intensity of two probe DNA-linked QDs was investigated at room temperature. As shown in Fig. 2, the fluorescence intensity of two QDs increases simultaneously with the increasing pH value from 5.0 to 10.0. It can be seen that green QDs (small size) are more susceptible than red QDs in different pH solutions. This phenomenon, reported by Maule and coworkers [28], could contribute to the result that the protonation/deprotonation process of the carboxylic ligands of the MPA may influence the QD bandgap energy. As the pH is increased and the carboxylic ligands ionize, a negatively charged shell builds up in the dots’ surface, which increases the confinement of the charge carriers in the semiconductor core and results in a higher bandgap energy, shorter emission wavelengths, and higher quantum yields [28]. The effect of ionic strength was also investigated by the addition of NaCl, as shown in Fig. 3. The result shows that fluorescence intensity of QDs decreases when the concentration of NaCl increased from 0 to 100 mmol L1. It can also be seen that green QDs are slightly more susceptible than red QDs in various levels of ionic strength. This is a result of the counter ion screening effect, which decreases the binding affinity of QDs with biomolecules [29,30]. Finally, pH in physiological condition (pH 7.4) was adopted, and no addition of NaCl was determined for the highest PL intensity in further experiments. Optimization of saturated volume of two QDs-labeled probe DNAs In this study, unreacted capture DNA on the surface of AMSMs must hybridize completely with complementary QDs-labeled probe DNA for the immunoassay. So, it is important to determine the optimized saturated added volume of QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA before target DNABRAF and target DNABRCA are introduced into the hybridization system. As shown in Fig. 4, the PL intensity of the hybridization system at 546 nm increases with increasing volumes of QDs546-labeled probe DNABRAF from 0 to 400 ll and then becomes flat when the added volume is more than 400 ll. Similarly, it can also be seen in Fig. 5 that the optimized saturated volume of QDs657-labeled probe DNABRCA is 400 ll. Therefore, 400 ll of QDs546-labeled probe DNABRAF and 400 ll of QDs657-labeled probe DNABRCA were used in further experiments.
Effect of pH and ionic strength In biological fluid, pH and ionic strength are important factors for the fluorescence of QDs. In this study, the effect of pH and ionic
Fig.1. (A) Normalized emission spectra of green CdTe QDs (solid line, Em 546 nm) and red CdTe QDs (dashed line, Em 657 nm). (B) Normalized fluorescence spectra of bare AMSMs without DNA (a), bare QDs incubated with capture DNA-modified AMSMs (b), QDs-labeled probe DNA incubated with capture DNA-modified AMSMs in the absence of target DNA (c), and QDs-labeled probe DNA incubated with capture DNA-modified AMSMs in the presence of target DNABRAF (20 nmol L1) and target DNABRCA (20 nmol L1) (d). The mass concentration of AMSMs in all experiments is 2.2 mg ml1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Detection of target DNABRCA and DNABRAF In the current work, the concentrations of target DNABRCA and DNABRAF can be detected simultaneously. It can be seen that when
Fig.2. Fluorescence intensity of two probe DNA-linked QDs in different pH environments (PBS, 10 mmol L1).
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Fig.3. Fluorescence intensity of probe DNA-linked two QDs in different levels of ionic strength. The concentration of added NaCl increases from 0 to 100 mmol L1.
Fig.4. Normalized fluorescence spectra of QDs546-labeled probe DNABRAF after incubation with capture DNABRAF-modified AMSMs for 6 h. Curves a to h present the added volumes of QDs546-labeled probe DNABRAF of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, and 0.8 ml, respectively. The concentration of capture DNABRAF and capture DNABRCA modified on the surface of AMSMs is 25 nmol L1.
two target DNAs exist in the reaction system, the PL intensity of dual-color QDs decrease together with the increasing concentrations of two target DNAs simultaneously (Fig. 6). This can be attributed to the mechanism of this work (see Scheme 1). When no target DNA was added, all of the capture DNA on the surface of AMSMs could hybridize completely with complementary QDs-labeled probe DNA. After the addition of target DNA, some capture DNA hybridized with target DNA and only the unreacted capture DNA on the surface of AMSMs could hybridize with complementary QDs-labeled probe DNA. The more target DNA added, the less unreacted capture DNA on the surface of AMSMs and the lower signals emitted. So, the PL intensity decreased with the increase of target DNA added in the current experiment. Under optimal conditions, we recorded the fluorescence emission spectrum change of the hybridization system after incubation with different concentrations of target DNABRAF. As shown in Fig. 7, the PL intensity of the hybridization system at 546 nm decreases gradually with increasing concentrations of target DNABRAF from 0.5 to 40 nmol L1. The inset in Fig. 7 displays the relationship between the PL intensity ratio (I/I0) and the concentration of target DNABRAF in the range of 0.5 to 25 nmol L1. I and I0 refer to the fluorescence
Fig.5. Normalized fluorescence spectra of QDs657-labeled probe DNABRCA after incubation with capture DNABRCA-modified AMSMs for 6 h. Curves a to h present the added volumes of QDs546-labeled probe DNABRAF of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, and 0.8 ml, respectively. The concentration of capture DNABRAF and capture DNABRCA modified on the surface of AMSMs is 25 nmol L1.
Fig.6. Fluorescence spectra of dual QDs-labeled AMSMs with different concentrations of target DNABRAF and target DNABRCA after incubation with capture DNABRAF and DNABRCA simultaneously on the surface of AMSMs for 6 h in solution. Curves a to d present the concentrations of target DNABRAF and target DNABRCA of 0, 5, 10, and 20 nmol L1, respectively.
intensity of the immunoassay system in the presence and absence of target DNABRAF, respectively. The linear regression equation was I/I0 = 0.99676-0.01062CBRAF (nmol L1), and the coefficient of correlation was R2 = 0.9963. The detection limit was 0.26 nmol L1 using the criterion of three times the standard deviation of the blank signal. This high DNA detection sensitivity can be attributed to the signal amplification of the nano-on-micro technique used in this study. It also can be seen in Fig. 8 that the PL intensity of the hybridization system at 657 nm decreases gradually with the increase of the concentration of target DNABRCA from 0 to 40 nmol L1. The inset in Fig. 8 displays the relationship between the PL intensity ratio (I/I0) and the concentration of DNABRCA in the range of 1 to 25 nmol L1. I and I0 refer to the fluorescence intensity of the immunoassay system in the presence and absence of target DNABRCA, respectively. The linear regression equation was I/ I0 = 0.99903-0.00939CBRCA (nmol L1), the coefficient of correlation was R2 = 0.998, and the detection limit was 0.57 nmol L1.
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Fig.7. Fluorescence spectra of dual QDs-labeled AMSMs with different concentrations of target DNABRAF hybridization with capture DNABRAF on the surface of AMSMs for 6 h in solution. Curves a to j present the concentrations of target DNABRAF of 0, 0.5, 1, 5, 10, 15, 20, 25, 30, and 40 nmol L1, respectively. The inset shows the relationship between the plot of I/I0 value and the concentration of target DNABRAF. I and I0 refer to the fluorescence intensity of the immunoassay system in the presence and absence of target DNABRAF, respectively.
Fig.9. (A) Relative fluorescence intensity (DI/I0, DI=I0-I) in DNABRAF detection in the presence of complementary target DNABRAF (a) and one-base mismatched DNABRAF (b). (B) Relative fluorescence intensity (DI/I0) in DNABRCA detection in the presence of complementary target DNABRCA (c) and two-base mismatched DNABRCA (d). I and I0 refer to the PL intensity of QDs in the presence and absence of target DNA or mismatched DNA in the hybridization system, respectively. The concentration of all the added DNA, including target DNA and mismatched DNA, is 25 nmol L1.
Table 2 Results for detection of BRAF and BRCA DNA simultaneously in human serum sample. DNA
Spiked (nmol L1)a
Found (mean ± RSD, n = 3)
Recovery (%) (mean ± RSD, n = 3)
BRAF
5 10 20
5.21 ± 0.12 9.72 ± 0.19 19.97 ± 0.24
104.3 ± 2.4 97.2 ± 1.9 99.9 ± 1.2
BRCA
5 10 20
5.05 ± 2.81 10.43 ± 1.91 19.33 ± 1.36
101.0 ± 2.8 104.3 ± 1.9 96.6 ± 1.4
a The original human serum samples were subjected to a 10-fold dilution in 1 ml of HB solution.
DNABRCA, respectively. These results indicate that the two mutant target DNAs both have lower hybridization ability than complementary target DNAs, which can attribute to the high selectivity of this hybridization system. Determination of DNABRAF and DNABRCA in real samples
Fig.8. Fluorescence spectra of dual QDs-labeled AMSMs with different concentrations of target DNABRCA hybridization with capture DNABRCA on the surface of AMSMs for 6 h in solution. Curves a to i present the concentrations of target DNABRCA of 0, 1, 5, 10, 15, 20, 25, 30, and 40 nmol L1, respectively. The inset shows the relationship between the plot of I/I0 value and the concentration of target DNABRCA. I and I0 refer to the fluorescence intensity of the immunoassay system in the presence and absence of target DNABRCA, respectively.
Selectivity of hybridization system In this study, the selectivity of this hybridization system was also investigated by using mutant DNA sequences. Fig. 9A shows the relative fluorescence intensity (DI/I0, DI=I0-I) in DNABRAF detection in the presence of complementary target DNABRAF (column a) and one-base mismatched DNABRAF (column b). Fig. 9B shows the relative fluorescence intensity (DI/I0) in DNABRCA detection in the presence of complementary target DNABRCA (column c) and twobase mismatched DNABRCA (column d). It can be seen that the DI/ I0 values obtained on the addition of 25 nmol L1 one-base mismatched DNABRAF and two-base mismatched DNABRCA are approximately 5.7% and 2.5% of the value obtained on the addition of the same concentration of complementary target DNABRAF and target
To evaluate the feasibility of the proposed method, the method was applied to the determination of DNABRAF and DNABRCA in human serum samples. The concentrations of DNABRAF and DNABRCA in samples were determined simultaneously, and the results are shown in Table 2. It can be seen that the quantitative recoveries of the spiked target DNA sequences are in the range of 96.6% to 104.3%. The relative standard deviation (RSD) was less than 2.81%. The results indicated that the accuracy and precision of the proposed method are satisfactory. All of these results indicate that the proposed method is a good multiplex DNA sensor for detecting target DNABRCA and target DNABRAF simultaneously. Conclusion In this study, a ‘‘nano-on-micro’’-based multiplex DNA detection optical sensor has been developed for simultaneous detection of DNABRAF and DNABRCA with dual-color fluorescence output signals in homogeneous solution. We demonstrated that the proposed method is effective for detecting DNABRAF and DNABRCA simultaneously with high sensitivity. The sensor has great potential to expand its application to the early diagnosis of cancers such as breast cancer, ovarian cancer, and PTC and even can detect other pathogenic DNAs by using different capture and target DNAs.
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Multiplex DNA sensor for BRAF and BRCA detection Xiangzhao Ai, Qiang Ma, Xingguang Su ⇑ Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 20 November 2012 Received in revised form 27 February 2013 Accepted 28 February 2013 Available online 13 March 2013 Keywords: Multiplex DNA sensor Quantum dots BRAF DNA BRCA DNA Simultaneous detection
a b s t r a c t In this article, a kind of simple, sensitive, and rapid quantum dots (QDs)-based multiplex DNA sensor is developed for the simultaneous detection of BRAF and BRCA DNA based on the ‘‘nano-on-micro’’ technique. In our strategy, capture DNABRCA and DNABRAF are simultaneously immobilized on the surface of amino-modified silica microbeads. After blocking with bovine serum albumin (BSA), different concentrations of target DNABRCA and DNABRAF are introduced to hybrid with complementary capture DNABRCA and DNABRAF. After hybridization, QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA were added into the above solution so that the unreacted capture DNABRCA and DNABRAF could be detected by QDs657-labeled probe DNABRCA and QDs546-labeled probe DNABRAF simultaneously. We demonstrate that the proposed method is effective for detecting BRAF and BRCA DNA with high sensitivity. The sensor has great potential to expand its application to the early diagnosis of cancers such as breast cancer, ovarian cancer, and papillary thyroid carcinoma. Ó 2013 Elsevier Inc. All rights reserved.
Recently, development of life science requires more sensitive and rapid analysis methods for various biomolecules, including protein, DNA, virus, and cells [1–4]. In these novel methods, optical techniques have been efficiently employed due to their fast, cheap, and high-throughput properties. Most optical analytical systems are based on various dyes, for example, cyanine-type and Alexa Fluor-type fluorophores [5]. Unfortunately, these organic dyes suffer from inherent disadvantages such as photobleaching, broad excitation and emission bands, small Stokes shift, and poor environmental stability. These properties significantly affect the uniformity of fluorescent signals and have some limitations for the trace analysis of biomolecules. Semiconductor quantum dots (QDs)1 are under intense attention based on their predominant optical properties. First, they have high quantum efficiency and a continuous absorption profile [6,7]. This allows different QDs emissions to be excited simultaneously using a single wavelength. Second, they have size-tunable emission spectra that make different-colored QDs (from blue to red) obtained by simple refluxing in different times [8]. Third, the resistance to photobleaching and high stability of QDs in the biological environment [9,10] permit QDs to be used in monitoring biological molecules in vivo. The advantages of QDs, ⇑ Corresponding author. E-mail address: [email protected] (X. Su). Abbreviations used: QDs, quantum dots; PTC, papillary thyroid carcinoma; AMSM, amino-modified silica microbead; BSA, bovine serum albumin; MPA, 3-mercaptopropyl acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; sulfo-NHS, N-hydroxysulfosuccinimide; TEOS, tetraethoxysilane; APTES, 3-aminopropyltrimethoxysilane; THPMP, 3-(trihydroxysilyl)-propylmethylphosphonate (THPMP); WB, washing buffer; HB, hybridizing buffer; PBS, phosphate buffer solution; PL, photoluminescence; RSD, relative standard deviation. 1
0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.02.029
such as narrow, symmetric, tunable emission spectra and large Stokes shift, enable them to be an excellent optical material for multiplex analysis. For instance, Freeman and coworkers reported on the use of T-rich aptamer and T-rich aptamer-modified QDs for the selective analysis of Hg2+ or Ag2+ ions using an electron transferquenching path [11]. Regarding the need for sensitive and multiple DNA detection in gene therapy and disease forecasting, many efforts have focused on the development of different DNA biosensors capable of measuring multiple targets in parallel. There are two broad classes of techniques used for multiplexing assay: planar arrays [12] and suspension arrays [13]. Different from conventional planar arrays, the advantages of suspension arrays include (i) enhanced reaction kinetics due to their suspension in homogeneous solution; (ii) the ability to measure analyte response to several different targets simultaneously; (iii) shorter incubation and assay time, leading to higher throughput; and (iv) higher sensitivity and lower cost. Thus, suspension arrays are suitable for multiplex assays and signal amplification for trace DNA detection. For instance, Jang and coworkers designed a high-sensitive multiplexed DNA detection assay using oligonucleotide-modified magnetic microparticle and gold nanoparticle probes that can simultaneously detect three kinds of DNA sequences in one sample [14]. Luo and coworkers developed a sensitive, magnetic, and multiplexed DNA detection biosensor for simultaneous detection of two target DNA sequences [15]. Owing to its unique properties, multiplexed DNA detection assay has shown great promise in clinical diagnostics. In this article, a novel, sensitive, and multiplexed DNA biosensor is proposed based on the ‘‘nano-on-micro’’ principle, which means
DNA sensor for BRAF and BRCA detection / X. Ai et al. / Anal. Biochem. 438 (2013) 22–28
that nanoparticles are conjugated on the surface of microbeads [16]. Compared with other methods, the nano-on-micro technique has two advantages in multiplex analysis and immunoassay for biomolecules. First, the large surface areas of microbeads make it possible to capture 100,000 analytes on 5.5-lm microbeads, which prominently improves the sensitivity for analysis [17]. In addition, analyte can be captured in a short time based on enhanced reaction kinetics of microbeads [18]. Here, we combine QDs with silica microbeads based on the nano-on-micro principle to develop a multicolor probe for simultaneous detection of two kinds of DNA: BRCA and BRAF. BRCA is a susceptive mutation DNA in human breast cancer that is responsible for most hereditary breast and ovarian cancers [19,20]. Moreover, it has been reported that missense BRAF mutation occurs in 29% to 83% of papillary thyroid carcinoma (PTC), which is the most common malignant tumor of the thyroid gland and the most frequent type of endocrine cancer [21]. Therefore, the detection of special base sequences in human genome such as BRCA and BRAF DNA is a crucial research topic for the effective treatment of malignant tumors, including breast cancer, ovarian cancer, and PTC, in the medical field. A few methods have been reported for the detection of BRCA and BRAF DNA. However, these methods have some inherent limitations. For instance, Li and coworkers reported a novel sandwich-type assay for the optical detection of BRCA DNA [22], but it has only a single signal emission that is hardly expanded to multiplexed DNA detection assay. Liao and coworkers presented a sensitive technique for the electrochemical detection of BRAF DNA [23], but it has an intricate mechanism and a convoluted electrochemical analysis procedure. Here, we propose a novel simultaneous detection strategy for BRCA and BRAF DNA based on the nano-on-micro technique, as shown in Scheme 1. First of all, capture DNABRCA (blue) and DNABRAF (purple) are simultaneously immobilized on the surface
23
of amino-modified silica microbeads (AMSMs) by glutaraldehyde. After blocking with bovine serum albumin (BSA), different concentrations of target DNABRCA (red) and target DNABRAF (dark green) are introduced to hybrid with complementary capture DNABRCA and DNABRAF for 10 h. After hybridization, QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA were added into the above solution so that the unreacted capture DNABRCA and DNABRAF could be detected by QDs657-labeled probe DNABRCA (red light emission) and QDs546-labeled probe DNABRAF (green light emission) simultaneously. To the best of our knowledge, this is the first report of the detection of BRCA and BRAF DNA simultaneously with the nano-on-micro principle. This novel sensor has great potential to expand its application to the early diagnosis of cancers such as breast cancer, ovarian cancer, and PTC. Materials and methods Materials and reagents All chemicals used were of analytical reagent grade without further purification. 3-Mercaptopropyl acid (MPA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) were purchased from J&K Chemical. Tellurium powder, CdCl2, NaBH4, Triton X-100, cyclohexane, n-hexanol, tetraethoxysilane (TEOS), 3-aminopropyltrimethoxysilane (APTES), 3-(trihydroxysilyl)-propylmethylphosphonate (THPMP), isopropyl alcohol, NaCl, and BSA were purchased from Sigma–Aldrich. The water used in all experiments had a resistivity higher than 18 MX cm. The oligonucleotides were purchased from Dingguo Biotechnology (Beijing, China) and purified using high-performance liquid chromatography. The sequences of oligonucleotides are shown in Table 1. In addition, washing buffer (WB, 10 mmol L1 Tris–HCl,
Scheme 1. Illustration of the mechanism of simultaneous detection of BRAF and BRCA DNA with dual-color QDs based on ‘‘nano-on-micro’’ technique.
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DNA sensor for BRAF and BRCA detection / X. Ai et al. / Anal. Biochem. 438 (2013) 22–28
pH 7.4), hybridizing buffer (HB, 10 mmol L1 Tris–HCl [pH 7.4] and 0.1 mol L1 NaCl), and phosphate buffer solution (PBS, 10 mmol L1, pH 7.4) were used in the whole following process. Instruments Fluorescence experiments were performed on an RF-5301 PC spectrofluorophotometer (Shimadzu, Japan), and a 1-cm pathlength quartz cuvette was used to measure the fluorescence spectrum. A bath ultrasonic cleaner (Autoscience AS 3120, Tianjin, China) was used to disperse the silica microbeads. All pH measurements were completed using an Ohaus STARTER 2100/3C meter (USA). An inverted fluorescence microscope (Olympus FV1000 IX71) equipped with a multispectral imaging system (Nuance, CRI, Woburn, MA, USA) was used to observe the fluorescence change of QDs. All optical measurements were carried out at room temperature under ambient conditions, and the excitation wavelength was 450 nm. Synthesis of AMSMs In the current study, AMSMs were prepared by the reverse microemulsion method at room temperature. In this method, cyclohexane was used as a continuous phase and Triton X-100 and n-hexanol were regarded as surfactant and cosurfactant, respectively. We mixed 15 ml of cyclohexane, 3.6 ml of Triton X100, 3.6 ml of n-hexanol, 800 ll of water, and 100 ll of TEOS to form a microemulsion system. After stirring for 30 min, 100 ll of NH4OH was added in solution to initiate the hydrolysis of TEOS. The reaction progressed in the dark with stirring for 24 h. Then, 30 ll of APTES and 80 ll of THPMP were injected into the reaction system, which was kept under stirring for 1 more day. Finally, the microemulsion was broken by adding 20 ml of acetone and the resultant precipitate was AMSMs, which were washed in sequence with ethanol and water several times. During each washing procedure, AMSM dispersion was first subjected to high-velocity centrifugation (10,000 rpm, 10 min), followed by decantation of the supernatant and redispersion of the precipitate in the next solvent with the aid of supersonication. Ultimately, aqueous AMSMs were dispersed in 1 ml of water and preserved in a refrigerator at 4 °C for further experiments. The mass concentration of AMSMs was 0.0548 g ml1. Preparation of CdTe QDs-labeled probe DNA CdTe QDs were synthesized by refluxing routes as described in detail in Ref. [24]. Briefly, the precursor solution of CdTe QDs was formed in water by adding fresh NaHTe solution to 1.25 103 mol L1 N2 saturated CdCl2 solution at pH 11.4 in the presence of MPA as stabilizing agent. The molar ratio of Cd2+/MPA/HTe was charged at 1:2.4:0.5. The CdTe precursor solution was subjected to reflux at 100 °C under open-air conditions with a condenser attached, and different sizes of CdTe QDs were obtained at different
refluxing times. The photoluminescence (PL) emission wavelengths of CdTe QDs used in this study were 546 and 657 nm. QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA were prepared by covalent link carboxyl and amino, respectively [25]. Briefly, 0.3 ml of 2.5 106 mol L1 EDC and 0.2 ml of 1.5 106 mol L1 sulfo-NHS were added into as-prepared 0.5 ml of QDs solution (Em 546 or 657 nm) to activate carboxyl of QDs with stirring for 0.5 h. Then, 100 ll of 1 106 mol L1 probe DNABRCA or probe DNABRAF was injected and stirred for 3 h to form a firm amide bond. Finally, 2 ml of isopropyl alcohol was added to precipitate QDs-labeled probe DNA, and unreacted reagents were removed by high-velocity centrifugation (10,000 rpm, 10 min). Ultimately, 1 ml of aqueous dispersed QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA was preserved in a refrigerator at 4 °C for further experiments. Preparation of capture DNA-modified silica microbeads In this study, capture DNABRCA and DNABRAF were linked with the amino groups on the surface of AMSMs by glutaraldehyde [26,27]. First, 40 ll of prepared AMSMs was suspended in 1 ml of PBS (2.2 mg ml1). Then, 100 ll of 1% glutaraldehyde was added to the solution, vibrating for 6 h. Remanent glutaraldehyde was removed by centrifugation, and the precipitation was redispersed in 1 ml of PBS by ultrasonic cleaner. Next, 25 ll of 1 106 mol L1 capture DNABRCA and 25 ll of 1 106 mol L1 capture DNABRAF were injected at room temperature and kept under stirring overnight. After that, 2 mg of NaBH4 was added, and the mixture was vibrated at room temperature for 2 h. The unreacted reagents were removed by centrifugation, and the precipitation was redispersed in 1 ml of PBS. Then, 1% BSA solution (10 mg ml1) was injected to block the unreacted sites on the surface of silica microbeads for 1 h, and remanent BSA was removed later. The precipitation was redispersed in 1 ml of HB solution by ultrasonic cleaner. Simultaneous detection of two target DNAs The immunoassay technique was used in this study to detect two target DNAs simultaneously. First, various concentrations of target DNABRCA and target DNABRAF were incubated with two capture DNA-modified AMSMs for 10 h at room temperature. Then, 400 ll of QDs546-labeled probe DNABRAF and 400 ll of QDs657-labeled probe DNABRCA were injected into the above solution and shaken overnight. Unreacted reagents were removed by centrifugation, and the precipitation was redispersed in 1 ml of HB solution after washing several times with WB solution. The PL intensity in this experiment was recorded with an excitation wavelength of 450 nm. For the real sample detection, the human serum samples were collected from healthy adult volunteers in a local hospital and deproteinized by centrifugation at 14,000 rpm for 10 min after adding acetonitrile in serum samples (CH3CN/serum, 1:1). Finally, all supernatant serum samples were subjected to a 10-fold dilution
Table 1 Sequences of oligonucleotides used in this study.
a
Name of oligonucleotide
Gene
Sequence of oligonucleotide (50 –30 )
Capture DNABRAF Probe DNABRAF Target DNABRAF One-base mismatched target DNABRCAa
BRAF BRAF BRAF BRAF
50 -GATTTTGGTCTAGCTACAGAGAAATCTCGA-(CH2)3-NH2-30 50 -TCGAGATTTCTCTGTAGCTAGACCAAAATC-(CH2)3-NH2-30 50 -TCGAGATTTCTCTGTAGCTAGACCAAAATC-30
Capture DNABRCA Probe DNABRCA Target DNABRCA Two-base mismatched target DNABRCAa
BRCA BRCA BRCA BRCA
50 -TCGAGATTTCACTGTAGCTAGACCAAAATC-30 50 -GAAACCCTATGTATGCTCTTTTTTTTTT-(CH2)3-NH2-30 50 -GAGCATACATAGGGTTTCTCTTGGTTTCTTTGATTATAATTCATAC-(CH2)6-NH2-30 50 -GAGCATACATAGGGTTTCTCTTGGTTTCTTTGATTATAATTCATAC-30 50 -GAGCATACATTGGGTTTCTCTTGGTTTCTTTGATTATTATTCATAC-30
The mutant nucleotide in the BRAF and BRCA mismatched target DNA is indicated in boldface and underlined.
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in 1 ml of HB solution, and different concentrations of target DNABRAF and DNABRCA were added into it to prepare the spiked samples. Results and discussion Fluorescence spectra of QDs and hybridization system Spectra overlap is a limiting factor in multiplex target detection using optical techniques. For the purpose of minimizing spectral interference, green CdTe QDs (Em 546 nm) and red CdTe QDs (Em 657 nm) were used in our experiment for labeling probe DNABRAF and probe DNABRCA, respectively. Fig. 1A shows the normalized emission spectra obtained from two kinds of CdTe QDs. It can be seen that the distance between the maximum emission peaks of two QDs is 111 nm, which is enough to detect two target DNAs without any spectral interference. Fig. 1B shows the normalized fluorescence spectra of bare AMSMs without DNA (curve a), bare QDs incubated with capture DNA-modified AMSMs (curve b), QDs-labeled probe DNA incubated with capture DNA-modified AMSMs in the absence of target DNA (curve c), and QDs-labeled probe DNA incubated with capture DNA-modified AMSMs in the presence of target DNA (curve d). It can be seen that the PL intensity of probe DNA-linked QDs (curves c and d) is much greater than that of bare QDs (curve b) after incubation with capture DNA modified on the surface of AMSMs, indicating that QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA can be successfully immobilized on the surface of AMSMs via DNA hybridization. In the absence of target DNA, as shown in curve c, all of the capture DNA on the surface of AMSMs could hybridize completely with complementary QDs-labeled probe DNA, so the PL intensity is the strongest. However, in the presence of target DNA, as shown in curve d, some capture DNA hybridizes with target DNA and only the unreacted capture DNA on the surface of AMSMs can hybridize with QDs-labeled probe DNA, so the PL intensity decreased. In addition, the weaker fluorescence intensity of bare QDs (curve b) after incubation with capture DNA modified on the surface of AMSMs is due to the effect of physical adsorption.
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strength on the PL intensity of two probe DNA-linked QDs was investigated at room temperature. As shown in Fig. 2, the fluorescence intensity of two QDs increases simultaneously with the increasing pH value from 5.0 to 10.0. It can be seen that green QDs (small size) are more susceptible than red QDs in different pH solutions. This phenomenon, reported by Maule and coworkers [28], could contribute to the result that the protonation/deprotonation process of the carboxylic ligands of the MPA may influence the QD bandgap energy. As the pH is increased and the carboxylic ligands ionize, a negatively charged shell builds up in the dots’ surface, which increases the confinement of the charge carriers in the semiconductor core and results in a higher bandgap energy, shorter emission wavelengths, and higher quantum yields [28]. The effect of ionic strength was also investigated by the addition of NaCl, as shown in Fig. 3. The result shows that fluorescence intensity of QDs decreases when the concentration of NaCl increased from 0 to 100 mmol L1. It can also be seen that green QDs are slightly more susceptible than red QDs in various levels of ionic strength. This is a result of the counter ion screening effect, which decreases the binding affinity of QDs with biomolecules [29,30]. Finally, pH in physiological condition (pH 7.4) was adopted, and no addition of NaCl was determined for the highest PL intensity in further experiments. Optimization of saturated volume of two QDs-labeled probe DNAs In this study, unreacted capture DNA on the surface of AMSMs must hybridize completely with complementary QDs-labeled probe DNA for the immunoassay. So, it is important to determine the optimized saturated added volume of QDs546-labeled probe DNABRAF and QDs657-labeled probe DNABRCA before target DNABRAF and target DNABRCA are introduced into the hybridization system. As shown in Fig. 4, the PL intensity of the hybridization system at 546 nm increases with increasing volumes of QDs546-labeled probe DNABRAF from 0 to 400 ll and then becomes flat when the added volume is more than 400 ll. Similarly, it can also be seen in Fig. 5 that the optimized saturated volume of QDs657-labeled probe DNABRCA is 400 ll. Therefore, 400 ll of QDs546-labeled probe DNABRAF and 400 ll of QDs657-labeled probe DNABRCA were used in further experiments.
Effect of pH and ionic strength In biological fluid, pH and ionic strength are important factors for the fluorescence of QDs. In this study, the effect of pH and ionic
Fig.1. (A) Normalized emission spectra of green CdTe QDs (solid line, Em 546 nm) and red CdTe QDs (dashed line, Em 657 nm). (B) Normalized fluorescence spectra of bare AMSMs without DNA (a), bare QDs incubated with capture DNA-modified AMSMs (b), QDs-labeled probe DNA incubated with capture DNA-modified AMSMs in the absence of target DNA (c), and QDs-labeled probe DNA incubated with capture DNA-modified AMSMs in the presence of target DNABRAF (20 nmol L1) and target DNABRCA (20 nmol L1) (d). The mass concentration of AMSMs in all experiments is 2.2 mg ml1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Detection of target DNABRCA and DNABRAF In the current work, the concentrations of target DNABRCA and DNABRAF can be detected simultaneously. It can be seen that when
Fig.2. Fluorescence intensity of two probe DNA-linked QDs in different pH environments (PBS, 10 mmol L1).
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DNA sensor for BRAF and BRCA detection / X. Ai et al. / Anal. Biochem. 438 (2013) 22–28
Fig.3. Fluorescence intensity of probe DNA-linked two QDs in different levels of ionic strength. The concentration of added NaCl increases from 0 to 100 mmol L1.
Fig.4. Normalized fluorescence spectra of QDs546-labeled probe DNABRAF after incubation with capture DNABRAF-modified AMSMs for 6 h. Curves a to h present the added volumes of QDs546-labeled probe DNABRAF of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, and 0.8 ml, respectively. The concentration of capture DNABRAF and capture DNABRCA modified on the surface of AMSMs is 25 nmol L1.
two target DNAs exist in the reaction system, the PL intensity of dual-color QDs decrease together with the increasing concentrations of two target DNAs simultaneously (Fig. 6). This can be attributed to the mechanism of this work (see Scheme 1). When no target DNA was added, all of the capture DNA on the surface of AMSMs could hybridize completely with complementary QDs-labeled probe DNA. After the addition of target DNA, some capture DNA hybridized with target DNA and only the unreacted capture DNA on the surface of AMSMs could hybridize with complementary QDs-labeled probe DNA. The more target DNA added, the less unreacted capture DNA on the surface of AMSMs and the lower signals emitted. So, the PL intensity decreased with the increase of target DNA added in the current experiment. Under optimal conditions, we recorded the fluorescence emission spectrum change of the hybridization system after incubation with different concentrations of target DNABRAF. As shown in Fig. 7, the PL intensity of the hybridization system at 546 nm decreases gradually with increasing concentrations of target DNABRAF from 0.5 to 40 nmol L1. The inset in Fig. 7 displays the relationship between the PL intensity ratio (I/I0) and the concentration of target DNABRAF in the range of 0.5 to 25 nmol L1. I and I0 refer to the fluorescence
Fig.5. Normalized fluorescence spectra of QDs657-labeled probe DNABRCA after incubation with capture DNABRCA-modified AMSMs for 6 h. Curves a to h present the added volumes of QDs546-labeled probe DNABRAF of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, and 0.8 ml, respectively. The concentration of capture DNABRAF and capture DNABRCA modified on the surface of AMSMs is 25 nmol L1.
Fig.6. Fluorescence spectra of dual QDs-labeled AMSMs with different concentrations of target DNABRAF and target DNABRCA after incubation with capture DNABRAF and DNABRCA simultaneously on the surface of AMSMs for 6 h in solution. Curves a to d present the concentrations of target DNABRAF and target DNABRCA of 0, 5, 10, and 20 nmol L1, respectively.
intensity of the immunoassay system in the presence and absence of target DNABRAF, respectively. The linear regression equation was I/I0 = 0.99676-0.01062CBRAF (nmol L1), and the coefficient of correlation was R2 = 0.9963. The detection limit was 0.26 nmol L1 using the criterion of three times the standard deviation of the blank signal. This high DNA detection sensitivity can be attributed to the signal amplification of the nano-on-micro technique used in this study. It also can be seen in Fig. 8 that the PL intensity of the hybridization system at 657 nm decreases gradually with the increase of the concentration of target DNABRCA from 0 to 40 nmol L1. The inset in Fig. 8 displays the relationship between the PL intensity ratio (I/I0) and the concentration of DNABRCA in the range of 1 to 25 nmol L1. I and I0 refer to the fluorescence intensity of the immunoassay system in the presence and absence of target DNABRCA, respectively. The linear regression equation was I/ I0 = 0.99903-0.00939CBRCA (nmol L1), the coefficient of correlation was R2 = 0.998, and the detection limit was 0.57 nmol L1.
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Fig.7. Fluorescence spectra of dual QDs-labeled AMSMs with different concentrations of target DNABRAF hybridization with capture DNABRAF on the surface of AMSMs for 6 h in solution. Curves a to j present the concentrations of target DNABRAF of 0, 0.5, 1, 5, 10, 15, 20, 25, 30, and 40 nmol L1, respectively. The inset shows the relationship between the plot of I/I0 value and the concentration of target DNABRAF. I and I0 refer to the fluorescence intensity of the immunoassay system in the presence and absence of target DNABRAF, respectively.
Fig.9. (A) Relative fluorescence intensity (DI/I0, DI=I0-I) in DNABRAF detection in the presence of complementary target DNABRAF (a) and one-base mismatched DNABRAF (b). (B) Relative fluorescence intensity (DI/I0) in DNABRCA detection in the presence of complementary target DNABRCA (c) and two-base mismatched DNABRCA (d). I and I0 refer to the PL intensity of QDs in the presence and absence of target DNA or mismatched DNA in the hybridization system, respectively. The concentration of all the added DNA, including target DNA and mismatched DNA, is 25 nmol L1.
Table 2 Results for detection of BRAF and BRCA DNA simultaneously in human serum sample. DNA
Spiked (nmol L1)a
Found (mean ± RSD, n = 3)
Recovery (%) (mean ± RSD, n = 3)
BRAF
5 10 20
5.21 ± 0.12 9.72 ± 0.19 19.97 ± 0.24
104.3 ± 2.4 97.2 ± 1.9 99.9 ± 1.2
BRCA
5 10 20
5.05 ± 2.81 10.43 ± 1.91 19.33 ± 1.36
101.0 ± 2.8 104.3 ± 1.9 96.6 ± 1.4
a The original human serum samples were subjected to a 10-fold dilution in 1 ml of HB solution.
DNABRCA, respectively. These results indicate that the two mutant target DNAs both have lower hybridization ability than complementary target DNAs, which can attribute to the high selectivity of this hybridization system. Determination of DNABRAF and DNABRCA in real samples
Fig.8. Fluorescence spectra of dual QDs-labeled AMSMs with different concentrations of target DNABRCA hybridization with capture DNABRCA on the surface of AMSMs for 6 h in solution. Curves a to i present the concentrations of target DNABRCA of 0, 1, 5, 10, 15, 20, 25, 30, and 40 nmol L1, respectively. The inset shows the relationship between the plot of I/I0 value and the concentration of target DNABRCA. I and I0 refer to the fluorescence intensity of the immunoassay system in the presence and absence of target DNABRCA, respectively.
Selectivity of hybridization system In this study, the selectivity of this hybridization system was also investigated by using mutant DNA sequences. Fig. 9A shows the relative fluorescence intensity (DI/I0, DI=I0-I) in DNABRAF detection in the presence of complementary target DNABRAF (column a) and one-base mismatched DNABRAF (column b). Fig. 9B shows the relative fluorescence intensity (DI/I0) in DNABRCA detection in the presence of complementary target DNABRCA (column c) and twobase mismatched DNABRCA (column d). It can be seen that the DI/ I0 values obtained on the addition of 25 nmol L1 one-base mismatched DNABRAF and two-base mismatched DNABRCA are approximately 5.7% and 2.5% of the value obtained on the addition of the same concentration of complementary target DNABRAF and target
To evaluate the feasibility of the proposed method, the method was applied to the determination of DNABRAF and DNABRCA in human serum samples. The concentrations of DNABRAF and DNABRCA in samples were determined simultaneously, and the results are shown in Table 2. It can be seen that the quantitative recoveries of the spiked target DNA sequences are in the range of 96.6% to 104.3%. The relative standard deviation (RSD) was less than 2.81%. The results indicated that the accuracy and precision of the proposed method are satisfactory. All of these results indicate that the proposed method is a good multiplex DNA sensor for detecting target DNABRCA and target DNABRAF simultaneously. Conclusion In this study, a ‘‘nano-on-micro’’-based multiplex DNA detection optical sensor has been developed for simultaneous detection of DNABRAF and DNABRCA with dual-color fluorescence output signals in homogeneous solution. We demonstrated that the proposed method is effective for detecting DNABRAF and DNABRCA simultaneously with high sensitivity. The sensor has great potential to expand its application to the early diagnosis of cancers such as breast cancer, ovarian cancer, and PTC and even can detect other pathogenic DNAs by using different capture and target DNAs.
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