A sensitive fluorometric DNA nanobiosensor based on a new fluorophore for tumor suppressor gene detection

Talanta 190 (2018) 140–146

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A sensitive fluorometric DNA nanobiosensor based on a new fluorophore for tumor suppressor gene detection

T

⁎

Maryam Darestani-Farahania, Farnoush Faridboda, , Mohammad Reza Ganjalia,b a b

Center of Excellence in Electrochemistry, School of Chemistry, College of Science, University of Tehran, Tehran, Iran Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Nanobiosensor DNA Tumor suppressor Gold nanoparticles Fluorescence Fluorophore

In this study, a sensitive fluorescent DNA nanobiosensor has been developed to determine DNA sequence of a well-known tumor suppressor gene, Adenomatous Polyposis Coli (APC). The design of the nanobiosensor was carried out using a synthetic organic ligand as a new fluorophore. The response mechanism of the nanobiosensor was based on DNA hybridization. The new fluorophore was assembled on gold nanoparticles (Au NPs) to enhance the sensitivity of the nanobiosensor response. The fabricated DNA nanobiosensor showed a fluorescence emission at 477 nm by exciting wavelength of 360 nm. By addition of the ssDNA target, the fluorescent emission of the nanobiosensor enhanced linearly in the range from 3.3 × 10−10 to 1.1 × 10−9 mol L−1 with detection limit of 1.3 × 10–11 mol L−1. The proposed DNA nanobiosensor responded selectively to its complementary strand in comparison with non-complementary and three mismatched bases. The nanobiosensor had also a fast response time with acceptable repeatability. Finally, the performance of the DNA nanobiosensor in biological fluid, serum plasma, was investigated and a satisfactory results were obtained.

1. Introduction Geneticists, for many years, have tried to find the reason of disease through detecting changes or mutations in genes. Study of human gene status can be a critical issue in diagnosis of many diseases such as diabetes, cardiovascular disease, autoimmune disorders, psychiatric illnesses and even cancer [1]. It is important in determining a successful treatment, early detection of tumors and improving the survival rates in the cancer infected patients [2]. Progress in DNA science and finding more about genes and their functions, make the analysis of genes at the molecular level possible. New technologies and analytical methods introduce day by day to find detection methods to be able to determine the changes in nucleic acids sequencing more sensitive and accurate [3]. In recent years, some methods have been used to determine the status of tumoric genes such as bisulphite genomic DNA sequencing, Southern blot, restriction enzyme-PCR, denaturing high-performance liquid chromatography (DHPLC) and electrochemical methods [4–8]. These methods need enzymes for specific modification of DNA bases [9,10] antibodies and proteins that bind to cytosine [11–14] or for some specific chemical changes [15,16] which are costly and timeconsuming. In the other hand, some DNA biosensors have been fabricated recently that are known as a great alternative method due to their ⁎

fast response, cost-effectiveness, being more sensitive and selective in addition to easy applications as a rapid test in decision making [17,18]. Currently, finding direct detection of DNA sequences without using chemical or enzymatic treatments is the area of interest for scientific communities and industries [19]. Designing a DNA biosensor based on fluorescent detection can be a good suggestion for its high sensitivity and high selectivity [20]. Also application of nanomaterials can improve the biosensors responses and may provide an inexpensive, rapid and easy to use analytical tools. Moreover, biosensors based nanomaterials are capable to be portable for point of care diagnosis [21]. To design a DNA biosensor, a synthetic organic ligand is used as a novel fluorescent DNA probe. It was selected from triazole thiol indole derivatives. In the other hand, selected ligand has interesting interaction with gold nanoparticles and has high photo-stability and quantum yield. The IUPAC name for the selected ligand is 4-((thiophen-2-yl) methyleneamino)-5-(1H-indol-3-yl)-4H-1,2,4-triazole-3-thiol. It is characterized by an intense π→π* transition absorption bands at 288 nm and 334 nm and a fluorescence at 477 nm. The investigation of ligand interactions with DNA is important to understand the mechanism of interaction and design new ligands [22]. Adenomatous Polyposis Coli (APC) is known as a tumor suppressor genes. It can inhibit the uncontrolled growth of cells which possibly be responsible for cancerous tumors. APC can be related with some

Corresponding author. E-mail addresses: [email protected], [email protected] (F. Faridbod).

https://doi.org/10.1016/j.talanta.2018.07.042 Received 5 January 2018; Received in revised form 11 July 2018; Accepted 12 July 2018 0039-9140/ © 2018 Elsevier B.V. All rights reserved.

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Scheme 1. Schematic representation of the designed DNA nanobiosensor for determination of APC gene sequence.

purchased from Sigma-Aldrich. Deionized water was used during the experiments. The Organic ligands was synthesized in the medical science University of Tehran, Tehran, Iran. The tested 24 bp oligonucleotides for the study were purchased from Shanghai Generay Biotech Co. The base sequences are as follow: Probe sequence (P): 5′-TCCGCTTCCCGACCCGCACTCCGC-3′; Target sequence (complementary sequence) (T): 5′-GCGGAGTGCG GGTCGGGAAGCGGA-3′; Three-base mismatched sequence (in CpG sites): 5′-GCCGAGTGCC GGTCGGGAAGCCGA-3′; Three-base mismatched sequence (in non-CpG sites): 5′-GCGGTGT GCGGGACGGGATGCGGA-3′; Non complementary sequence: 5′-CTTATCCTTTAGTTTATGTCT TAT-3′. All oligonucleotides stock solutions were made with TE Buffer and kept in refrigerator before use. 0.01 mol L−1 Tris–HCl solution (in pH 8) and 0.001 mol L−1 EDTA solution were used to make a TE Buffer. Dilute solutions were prepared with 0.01 mol L−1 Tris-HCl buffer containing 0.5 mol L−1 NaCl in pH 7.4.

cellular routes, such as apoptosis, cell-cycle regulation, cell adhesion, cell migration, microtubule assembly and cell fate determination [23]. Mutations in the APC gene have been seen in about 80% of all human colon cancers. Thus, it can be used as a marker for early diagnosis of cancerous transformations [24]. Here, due to high importance of determining this gene, a specific sequence of APC gene previously reported [19] was selected as the target molecule. In this research, we introduced a new fluorophore as a fluorescent probe in fabrication of a DNA biosensor that can determine the concentration of APC gene in human plasma samples. Fluorophore molecules are immobilized on the gold nanoparticle to improve the sensitivity and then ssDNA probe is added to the solution. The response mechanism of the nanobiosensor was based on DNA hybridization. As illustrated in Scheme 1, the addition of ssDNA target results in gradual increase of the emission. The fabricated optical DNA nanobiosensor has fast response, with good selectivity and sensitivity. 2. Experimental details 2.1. Instruments and measurements All fluorescence experiments were recorded with a Perkin Elmer LS45 fluorescence spectrometer (UK). Ultra violet- visible (UV–Vis) spectroscopy was performed using a Specord 250 spectrophotometer (Germany) and Infra-Red (IR) spectra were recorded using a BrukerTensor 27.

2.3. The synthesis of organic ligands The general procedure for the preparation of the ligands was done through a well-known Schiff's Base reaction [25]. The general procedure was as follow: a mixture of a (0.01 mol) X compound (In ligand I, X is Thiophen-2-carbaldehyde; ligand II, Pyrrol-2-carbaldehye and ligand III, Furan-2-carbaldehyde) with 4-amino-5-(1H-indol-3-yl)-4H-1,2,4triazole-3-thiol (0.01 mol) and a catalytic amount of acetic acid was refluxed for 5 h in absolute ethanol (20 mL). Then the solvent was evaporated to 5 mL, cooled to room temperature, and the product was

2.2. Materials All chemicals used were purchased from Merck Co. (Germany). Trishydrochloric acid (Tris-HCl) and chloroauric acid (HAuCl4·4H2O) were

Fig. 1. (a) General synthesis of the ligand I, II and III (In ligand I, X is Thiophen-2-carbaldehyde; ligand II, Pyrrol-2-carbaldehye and ligand III, Furan-2-carbaldehyde) (b) TEM image of synthesized Au NPs. 141

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filtered, washed with ethanol and dried under the reduced pressure (Fig. 1a). The purity of obtained organic products were confirmed by nuclear magnetic resonance spectroscopy (1H NMR,13C NMR, Fig. S1).

2.7. Sample preparation of the human plasma A drug free human plasma was gotten from the Iranian Blood Transfusion Service (Tehran, Iran) and kept at −20 °C. To precipitate the plasma proteins, the samples were treated with 1 mL nitric acid 0.1 mol L−1. After that, the mixture was vortexed for a 30 s and then centrifuged at 12,000 rpm for 10 min and then the upper plasma solution transferred to a clean vial. Then, 100 μL aliquot of the obtained supernatant was diluted 10 times and used for analyzing.

2.4. Synthesis of Au NPs Fifty milliliters aqueous solution of hydrogen tetrachloroaurate(III) tetrahydrate (0.001 mol L−1) was heated to boil while being stirred in a round-bottom flask with a reflux condenser. Then 10 mL of trisodium citrate (0.0388 mol L−1) was added into the solution rapidly and the solution was boiled for another 10 min while the color of the solution changed from yellow to darkish red. After that, the heating was stopped and the solution was continued stirring to cool down to the room temperature [26], then the Au NP solution was stored in a refrigerator at 4 °C. According to Beer's law by using the absorption coefficient (2.7 × 108 L mol−1 cm–1) at 520 nm [27], the concentration of the Au NP solution was obtained 4.4 × 10−9 mol L−1 and they had a diameter about 13 nm (Fig. 1b).

3. Results and discussion 3.1. The fluorophore selection Initially, studies were done on three synthesis organic ligands that they were from one family and their structures were similar to bases of DNA, hence it is predicted they can react with them (Fig. 3a). At first solutions of each ligand were prepared in optimized conditions of each ligand to show acceptable fluorescence emission, while they had different solubility in water, different concentrations and different excitation and emission wavelength (Fig. 3b,c). Results showed that optimized solubility of ligand I was 70/30 methanol/water in concentration 5 × 10−5 mol L−1 and its maximum wavelength of excitation and emission was 360 nm and 477 nm, respectively (red curves in absorbance and emission spectra in Fig. 3b,c. Ligand II solved in 50/ 50 methanol/water, 10−4 mol L−1 and its maximum wavelength of excitation and emission was 330 nm and 490 nm, respectively (blue curves in absorbance and emission spectra in Fig. 3b,c). Also optimized condition about ligand III was solubility in 90/10 methanol/water, 10−3 mol L−1 and its maximum wavelength of excitation and emission was 320 nm and 490 nm, respectively (green curves in absorbance and emission spectra in Fig. 3b,c). From these studies can be found that solubility and fluorescence intensity of ligand I is better than ligand III. Also the fluorescence intensity of ligand I is higher than ligand II. Because of existence S-H in structure of ligands, they are able to connect on gold nanoparticles, therefore they were tested to investigate their response to Au NPs. After studying the solubility and optical properties of ligands, 10 μL of Au NPs was added to each ligand solution in optimized conditions and their fluorescence emission changes are demonstrated by molecular fluorescence spectroscopy (Fig. 3c). As it can be seen, Au NPs has significantly effect on intensity fluorescence signal of ligand I in compare to other ligands. The reason for this behavior is the extreme tendency of sulfur atom to interact with gold (III) [31] and presence of two sulfur atoms in structure of ligand I that it result in immediate self-assembly reaction with covalence binding and turn to be more rigidified. Therefore, the intensity of fluorescence signal was increased strongly [20]. The above investigations helped us to choose ligand I as a fluorophore for fabricating DNA biosensor, due to its high fluorescence

2.5. Preparation of DNA nanobiosensor Initially, solution of fluorophore was prepared with 5 × 10−5 mol L−1 concentration in 30–70 water-methanol solvent (Fig. 2, step 1). Then 10 μL of Au NPs was added to 3 mL of fluorophore in fluorescence cell, pipetting was done and 5 min was given to bind gold nanoparticles and sulfur atoms of fluorophore completely (Fig. 2, step 2). In the next step, 5 μL of ssDNA probe with 10−6 mol L−1 concentration was added to the cell containing fluorophore and Au NPs. DNA probe strands were subjected to a preheat treatment before use. For this purpose, the DNA solution was heated at 90 °C for 2 min and then left at room temperature to 50 °C [28,29]. After adding the ssDNA probe to incubate and complete interaction between fluorophore and ssDNA probe, it was shaken to mix and complete interaction between fluorophore and bases of ssDNA by vortex 1200 rpm for 10 min (Fig. 2, step 3). 2.6. Fluorescence measurements Hybridization process was done by gently stirring at 37 °C. However, DNA target strands were also subjected to a preheat treatment before use. It was heated at 90 °C for 2 min and then left at room temperature to 50 °C [28,29]. At first 20 μL of ssDNA target with 5 × 10−8 mol L−1 concentration was added to prepared DNA nanobiosensor in fluorescence cell and placing in Ben Murray at 37 °C for 30 min, as optimized hybridization time [30]. Titration was continued with addition of extra 10 μL in each step and performing repetitive process for each step. Fluorescence determination of the DNA hybridization was done at excitation and emission wavelength of 360 nm and 477 nm respectively (Fig. 2, step 4).

Fig. 2. Sequence of preparing DNA nanobiosensor (step 1–3) and its response to ssDNA target (step 4). 142

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Fig. 3. (a) Molecular structure of three synthetic ligands (b) UV–Vis Spectra of the ligands in optimized conditions of each ligand (c) Fluorescence spectra of the ligands in optimized conditions of each ligand and the effect of gold nanoparticles addition on them.

3.2. Immobilization of the fluorophore on gold nanoparticles As it mentioned in previous section, according to the structure of the fluorophore, sulfur atoms interacted with Au NPs. The self-assembly reaction with covalent bind is caused the structure of fluorophore became more rigid, therefore intensity of fluorescence emission enhanced (Fig. 2, step 2). The enhancement of the fluorescence can be attributed to a kind of photoinduced electron transfer (PET) (Lakowicz and Masters 2008). PET is an excited state electron transfer process by which the excited electron is transferred from donor to acceptor. Using surface science techniques, it has been proven that sulfur is adsorbed on S2-, SH- and SH2 in aqua electrolytic solutions and S2 and SO2 in gas phase. In all cases, sulfur is adsorbed by forming strong covalence bind on Au(III). Therefore, if Au NPs are used instead of Au (III) sulfur atoms in fluorophore structure will have more interaction with Au particles due to expanding the surface [32]. To optimization of Au NPs amount, different concentration of Au NPs in solution (7.3 × 10–12 – 2.9 × 10–11 mol L−1) was tested. It has been observed that as Au NPs concentration in solution increase, enhancement of fluorescence intensity increase too and it was favorable because the sensitivity is improved but due to the limitation of fluorescence spectrometer, 1.5 × 10–11 mol L−1 is chosen as an optimized concentration. This reaction was investigated by FT-IR spectra. As it is showed on Fig. 4, by addition of Au NPs to fluorophore solution, peaks of R-S-H in 2950–2750 cm−1 is almost omitted, which indicates the interaction between sulfur atoms and Au NPs. The peaks related to S-H bonds are demonstrated in upper wavelength rather than range of wavelength in the literature that it is effect of presence electronegative atoms near S atoms. The aim of using Au NPs in fabricating the DNA biosensor is immobilization of flourophore and improvement of sensitivity. As well when fluorophore immobilized on gold nanoparticles, dynamic quenching effect decrease, thus repeatability of method is improved.

Fig. 4. FT-IR spectra of fluorophore (blue spectrum), fluorophore immobilized on Au NPs (orange spectrum), fluorophore immobilized on Au NPs and interaction with ssDNA probe (green spectrum) and presence of ssDNA target in solution (red spectrum).

emission and sensitivity improvement of the DNA biosensor. The quantum yield (ϕ) of the ligand I was calculated using quinine sulfate (ΦR = 0.55) as the reference standard. The quinine sulfate was dissolved in 0.1 mol L−1 H2SO4 (refractive index (η) of 1.33) and the ligand was dissolved in methanol/water (η = 1.33).

ϕ = ϕR ×

η2 I A × R × 2 IR A ηR

Where Φ is the quantum yield, I is the measured fluorescence emission intensity, η is the refractive index of the solvent, and A is the absorbance at the excitation wavelength. The subscript R refers to the reference fluorophore of known quantum yield. The Φ value of the ligand was found to be 0.16.

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was reduced during the hybridization process which caused an increase in fluorescence emission. Indeed stable structure of helix DNA was formed with less flexibility than previous status [22]. Therefore, during the hybridization process, the number of hydrogen bonds between fluorophore and probe ssDNA have been reduced, as it has clearly been shown on FT-IR spectra (Fig. 4). The reduction in the intensity of N-H peaks shows the number of hydrogen bonds have been decreased in presence of ssDNA target. 3.5. DNA biosensor optimization pH and hybridization time as two important parameter which affects the biosensor response were studied (Fig. 7). Fig. 7a shows the DNA biosensor is extremely depended on pH due to the structure of fluorophore. In alkaline environments, fluorescence emission intensity significantly increased and overloaded, while in acidic environments, the fluorescence is extremely decreased until quenching completely. Furthermore, according to the Fig. 7b, increasing the incubation time for one hour caused a better responses. Based on the obtained results, the pH of 5.8 and the hybridization time of 30 min was used for next experiments.

Fig. 5. Fluorescence spectra of fluorophore immobilized on Au NPs in the presence of different concentration of ssDNA probe, Inset: logarithmic plot for fluorescence intensity ratio versus concentration of ssDNA probe (Stern-Volmer curve).

3.3. Interaction of fluorophore immobilized on AuNPs with ssDNA probe 3.6. Selectivity study In the next step, a sensible quenching was seen by addition of probe ssDNA with 10−6 mol L−1 to the cell containing fluorophore and Au NPs (Fig. 2, step 3). For having a better understanding about the mechanism of quenching of fluorescence emission, specified volumes of probe ssDNA with 10−6 mol L−1 concentration was titrated to the cell containing fluorophore and Au NP. Then, stern-volmer curve was drawn (Fig. 5). It followed of linear equation with regression coefficient (R) 0.9906. It shows the quenching is static type. Thus, fluorophore and bases of probe ssDNA bind to each other and form stable complex. According to the regression equation of Stern-Volmer curve, F0/F = 2 × 108C + 1.0897 (unit of C is mol L−1), which clearly results in a large number for Ksv. Hence we found out probe ssDNA have located on a surface of fluorophore which is in agreement with the other reported works [20]. FT-IR spectroscopy was used to confirm this reaction is taken place. Wide bond of N-H peak reveals the formation of hydrogen bond between fluorophore and bases of probe ssDNA (Fig. 4). Furthermore molecular fingerprint of pure DNA lies in the spectral region of 1800–700 cm−1 due to the plane vibrations of various nitrogenous bases, phosphate stretching vibrations (asymmetric and symmetric) and deoxyribose stretching [33].

To study the specificity and selectivity of the DNA biosensor, designed DNA biosensor was hybridized with different sequences included (A) non-complementary sequence, (B) three-base mismatched sequence (in CpG sites), (C) three-base mismatched sequence (in non-CpG sites) and (D) complementary sequence with the same concentration (6.5 × 10−10 mol L−1) and the related data were compared (Fig. 8). Fluorescence intensity of hybridization with non-complementary sequence showed a slight reduction due to dilution. Moreover, low fluorescence emission were found for hybridization with three-base mismatched target sequences in comparison with complementary target. The results showed the high selectivity of DNA biosensor and revealed that unspecific targets could not interfere. Moreover, the results showed three-base mismatched sequence (in CpG sites) had less interaction with ssDNA probe than three-base mismatched sequence (in non-CpG sites). Hence, as was expected, this data showed that interaction between fluorophore and CpG islands was stronger than other bases. 3.7. Plasma sample analysis Finally, the proposed biosensor, was successfully applied in real sample condition. The experiments were repeated in the human blood plasma instead of Tris-HCl buffer. APC gene sequence with five different concentrations (from 3.50 × 10−10 to 9.50 × 10−10 mol L−1) were spiked into the plasma sample vials and they were added to the prepared DNA biosensor in five falcons (samples number 1–5) and detection procedure was done based on the method described earlier in the fluorescence detection section. The results were acceptable and showed recoveries between 94.22% and 104.60% as illustrated in Table 1. Therefore, the results confirmed that DNA biosensor could be used for APC gene detection in plasma matrixes.

3.4. Influence of ssDNA target sequences on fabricated DNA nanobiosensor The response of the fabricated DNA biosensor is based on the enhancement of the fluorescence emission (Fig. 2, step 4). As it has been shown in Fig. 6a, upon the addition of ssDNA target, enhancement of the emission was observed. Response range of the DNA biosensor and calibration curve of fluorescence emission changes versus concentration of target ssDNA have been shown on Fig. 6b. Detectable and measurable linear range of the fabricated DNA biosensor was 3.3 × 10−10 –1.1 × 10−9 mol L−1 with regression equation F /F0 = 4×108C + 0.9406 (unit of C is mol L−1), determination coefficient (R2) was 0.9955 and its detection limit was calculated 1.3 × 10–11 mol L−1. As it was mentioned in the previous section, bases of the probe ssDNA formed hydrogen bond with immobilized fluorophore on Au NPs. After the interaction, new vibration levels of energy was created for the fluorophore, therefore non-radiative relaxation increased, which caused the quenching of the fluorescence emission. By entering the target ssDNA, since the probe ssDNA had stronger tendency to be hybridized with the target, it separated from the fluorophore. Hence, it can be proposed that the previous available vibration levels of the fluorophore gradually were omitted and intersystem crossing process

3.8. Validation of the biosensor The proposed biosensor was finally validated in terms of linearity, detection limit (LOD and LOQ), selectivity, precision (repeatability intra-day and inter day), reproducibility and robustness. As mentioned above, the linear measuring range of the proposed DNA biosensor was 3.3 × 10−10 to 1.1 × 10−9 mol L−1 with LOQ of 3.3 × 10−10 and LOD of 1.3 × 10–11 mol L−1 (equal to 0.0001 μg/mL for 24-pair base sequence). The proposed biosensor could selectively recognize the APC sequence from non-complementary sequences (RSD less than 3.35%). 144

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Fig. 6. (a) Fluorescence spectra of fabricated DNA biosensor in the presence of different concentration of ssDNA target, (b) Logarithmic plot for fluorescence intensity ratio versus concentration of ssDNA target: 1.6 × 10−10, 3.3 × 10−10, 4.9 × 10−10, 6.5 × 10−10, 8.2 × 10−10, 9.8 × 10−10, 1.1 × 10−9, 1.3 × 10−9, 1.4 × 10−9 mol L−1.

Fig. 7. (a) Effect of pH on fluorescence intensity of fabricated DNA biosensor (b) Effect of hybridization time on the fluorescence intensity of fluorophore (concentration of fluorophore: 5 ×10−5 mol L−1). Table 1 Recovery test for real sample of human blood plasma. Sample

Added DNA (mol L−1)

Detected DNA (mol L−1)

No. No. No. No. No. No. No. No. No. No.

3.50 × 10−10 0 5.00 × 10−10 0 6.50 × 10−10 0 8.00 × 10−10 0 9.50 × 10−10 0

3.31 NDb 5.23 ND 6.12 ND 8.30 ND 9.17 ND

a b

Fig. 8. Histogram of the fluorescence intensities for different DNA sequences, (a) non-complementary sequence, (b) three-base mismatched sequence in CpG sites (c) three-base mismatched sequence in non-CpG sites and (d) full complementary sequence (concentration of ssDNA sequences: 6.5 × 10−10 mol L−1).

1 1 2 2 3 3 4 4 5 5

± 0.05 × 10−10a −10

± 0.07 × 10

± 0.05 × 10−10 ± 0.6 × 10−10 ± 0.05 × 10−10

Recovery (%) 94.57 – 104.60 – 94.22 – 103.75 – 96.52 –

The results are based on triplicate measurements. Not detected.

examined while some parameter values such as pH of the test solution, hybridization time and laboratory temperature were slightly changed (about 5%). The obtained results were good and did not show any significant change when the critical parameters were changed (RSD less than 3.71%).

The obtained RSD values for the intra-day assays of 5 replicates at three different concentrations of APC gene sequence in the same laboratory condition by a same operator did not exceed 3.12%. The RSD% for inter-day precision, calculated by the analysis of five replicates standard samples on three consecutive days was 3.86%. Reproducibility of the proposed biosensor was also studied. RSD% in the response of five replicate biosensors which were prepared with a similar method and in the same laboratory conditions was 4.26%. Finally, robustness was

4. Conclusion In this study, we introduced a novel fluorophore (4-((thiophen-2-yl) methyleneamino)-5-(1H-indol-3-yl)-4H-1,2,4-triazole-3-thiol) for fabricating fluorescence DNA biosensor due to its high quantum efficiency, good emission wavelength and usability in the aquatic environment. Assays were also done for determination of APC gene based on novel fluorimetric DNA biosensor. Initially, fluorophore molecules were 145

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immobilized on gold nanoparticles for increasing repeatability and sensitivity of DNA biosensor that it was a new methodology in fabricating DNA biosensors. In next step, by considering the interaction between fluorophore and bases of ssDNA probe via fluorescence and FTIR spectra, it was proven that hydrogen bond was formed between fluorophore and bases of ssDNA probe. When ssDNA target sequence was added to prepared DNA biosensor, the fluorescence emission enhanced with increase in target ssDNA concentration. The changes in fluorescence intensity were linear with the concentration of ssDNA (3.3 × 10−10 – 1.1 × 10−9 mol L−1) and detection limit were 1.3 × 10–11 mol L−1. Finally, this DNA nanobiosensor showed a simple, sensitive and rapid method to determine the concentration of APC gene sequence in the real plasma samples.

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Acknowledgments [17]

The authors are grateful to the Research Council of University of Tehran for the financial support of this work.

[18]

Appendix A. Supporting information

[19]

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.talanta.2018.07.042.

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