A Highly Sensitive Electrochemical DNA Biosensor from Acrylic-Gold Nano-composite for the Determination of Arowana Fish Gender

A Highly Sensitive Electrochemical DNA Biosensor from Acrylic-Gold Nano-composite for the... The present research describes a simple method for the identification of the gender of arowana fish (Scleropages formosus). The DNA biosensor was able to detect specific DNA sequence at extremely low level down to atto M regimes. An electrochemical DNA biosensor based on acrylic microsphere-gold nanoparticle (AcMP-AuNP) hybrid composite was fabricated. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesised with a facile and well-established one-step photopolymerization procedure and physically adsorbed on the AuNPs at the surface of a carbon screen printed electrode (SPE). The DNA biosensor was constructed simply by grafting an aminated DNA probe on the succinimide functionalised AcMPs via a strong covalent attachment. DNA hybridisation response was determined by differential pulse voltammetry (DPV) technique using anthraquinone monosulphonic −18 acid redox probe as an electroactive oligonucleotide label (Table 1). A low detection limit at 1.0 × 10 M with a −18 −8 2 wide linear calibration range of 1.0 × 10 to 1.0 × 10 M(R = 0.99) can be achieved by the proposed DNA biosensor under optimal conditions. Electrochemical detection of arowana DNA can be completed within 1 hour. Due to its small size and light weight, the developed DNA biosensor holds high promise for the development of functional kit for fish culture usage. Keywords: DNA biosensor, Electrochemical biosensor, Acrylic microspheres, DNA hybridization, Photopolymerization, Arowana DNA Background good luck and happiness, along with many other cultures Asiatic arowana (Scleropages formoss), a freshwater fish, [1] [6]. Generally, the arowana is around 7 kg weight and 1 m is widely distributed over the countryside of Southeast Asia long in their mature age [9]. This ornamental fish possesses region such as Malaysia, Singapore, Thailand, Indonesia, attractive colours and morphology and can be identified by Cambodia, Vietnam, Laos, Myanmar and the Philippines. its distinctive physical features, such as comparatively long In addition, the arowana fish is also found in Australia and in body size, a large pectoral fin, and the dorsal and anal New Guinea [1–4]. It is popularly known as dragonfish, fins are positioned far back on the body. There are three Asia bonytongue, kelisa, or baju-rantai [5, 6]. It is still sur- main colour varieties, i.e. golden, red, and green of closely viving as a primitive fish species from the Jurassic era [7, 8]. related freshwater fish within the Asian arowana species. The Chinese and Asian people considered it as a symbol of There are also several other distinct species derived from different parts of the Southeast Asia and are regional to many river systems [8]. * Correspondence: mahbub_iuchem@yahoo.com Department of General Educational Development (GED), Faculty of Science Due to its high popularity and great demand in ornamen- & Information Technology, Daffodil International University, Dhanmondi, tal purposes, Asian arowana has been fiercely hunted for Dhaka 1207, Bangladesh profits [6], and results in a rapid decline of its population. School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Considering its high demand in ornamental industry, the Darul Ehsan, Malaysia over-exploitation of natural populations, and the rarity of Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 2 of 10 Table 1 Sequences of oligonucleotides utilised in the present the attempt to identify the gender and colour at their early investigation age. Traditional method based on body size and mouth DNA Base sequences cavity estimations can only be made at around 3 months of age of baby arowana for gender and colour identifica- DNA Probe 5'-AAT TCA AGG GAA CTG ATG ACT CTA (AmC7) tions [17]. However, this conventional visual examination cDNA 5'-TAG AGT CAT CAG TTC CCT TGA ATT method is time-consuming and often provides inaccurate ncDNA 5'-CGA GCG ACG TGA GCT TAG CTG CGC result. On the other hand, the widely used standard methods based on DNA sequencing, i.e., polymerase chain natural habitats due to changes in the living environment, reaction (PCR) and gel electrophoresis are labour-, time-, Asian arowana has been classified as an endangered species and resource-demanding. An alternative algorithm of in- threatened with extinction since 1980 by the Convention ventive problem solving (ARIZ) method was previously on International Trade in Endangered Species of Wild employed for the detection of arowana gender detection Faunaand Flora(CITES) andhas recently listed as endan- [18]. ARIZ is an alternative tool for gender detection, con- gered by the 2006 IUCN Red List [1, 3, 8, 10, 11]. However, taining nine different parts and a total of 40 complex the commercial trading of this endangered species is pro- steps. It requires a very long time to learn and practice hibited under CITES except in certain countries, e.g., and demands highly experienced personnel to operate. Indonesia, Singapore, and Malaysia. [2, 3, 12]. There are a For example, the application of ARIZ in various engineer- number of CITES registered cultivators in Asia actively car- ing systems has been employed, but most of the cases did rying out farming and trading of arowana fish [2, 12]. This not cover all the requirements and processes of ARIZ. Asian freshwater fish consists of geographically isolated In this research, acrylic polymer microspheres strains, and it is the only member of the species with differ- modified with succinimide functional groups via N- ent colour varieties that is based on different geographic acryloxysuccinimide (NAS) moieties was used as the distributions throughout the rivers of Southeast Asia. The matrix for DNA probe immobilisation. As previously re- species distribution is now far more widespread, which ex- ported by Chen and Chiu 2000 and Chaix et al. 2003 tends to the Nile River of Africa, the Amazon River of [19, 20], the succinimide functional group can react with South America, Australia, and New Guinea [1, 4, 8]. amine functional groups to form a covalent bond. The Among the different colours of Asiatic arowana, red and incorporation of NAS functionality into acrylic micro- golden arowana fishes are the most expensive and popular spheres for DNA microbiosensor application provides ornamental pets in the hatchery industry compared to advantages of a simple preparation method where the black, green, silver, and others colour varieties [1, 5, 10, 13]. spheres can be synthesised and functionalised via a one- The egg thievery phenomenon of Asiatic arowana is atyp- step procedure using photopolymerisation in a short ical compared to other fish species. In general, arowana duration (several minutes). In addition, the microspheres fishes get mature at the age of 3–4 years, and they lay only have the advantage of small size and provide a large sur- afew eggs (30–100) [14, 15] of extra-large size (around face area for DNA probe immobilisation, thus reducing 1 cm in diameter) [16]. Interestingly, the fertilised eggs and the barrier to diffusion for reactants and products. This larvae are then protected and grown up in the mouth of enables the improvement in the biosensor performance male arowana fishes, and they show high parental care. To in terms of shorter response times and wider linear re- identify the gender based on visual observation of the baby sponse range, which will be demonstrated in the work arowana is difficult because there is no distinctive pheno- reported here. typic organ of sexual dimorphism [14, 15]. Only one of the In this study, an electrochemical DNA biosensor parents (presume to be the male) can be identified as the method, which is highly sensitive, simple, easy-to- offspring are harvested from his mouth. The other parent fabricate, and low cost, is proposed for juvenile arowana cannot be identified from among a number of potential fish gender determination with high accuracy. The DNA parents [16]. biosensor was built from a carbon screen printed elec- Usually, the hobbyists keep the baby arowana fish for trode (SPE) modified with colloidal gold nanoparticles their ornamental purposes in the aquarium as well as for (AuNPs) and polyacrylate microspheres functionalised cultivation in the fish farm. However, all types of juvenile with NAS functional group. The AuNPs were immobilised arowana fishes are sold at the same price, because of the onto the carbon SPE surface via electrostatistic interaction lack of assistive technology for gender and colour variety and played an important role in enhancing the electrode differentiation. Until the present time, there is no estab- conductivity and facilitating the electron transfer, while lished method published to identify the gender and colour the acrylic microspheres (AcMPs) were directly deposited of arowana fishes at their juvenile stage. Instead, hundreds onto the AuNP-modified SPE via physical adsorption. of studies have been carried out using DNA analysis based Aminated DNA probe of arowana was then covalently at- on genetic structure and biography of arowana fishes in tached to the immobilised AcMP-AuNP composite at the Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 3 of 10 exposed succinimide group of AcMPs. Probe-target hy- Fabrication of DNA Biosensor Using Acrylic Microspheres bridisation was detected with anthraquinone redox label Prior to surface modification, the carbon SPE was rinsed via differential pulse voltammetry (DPV). The incorpor- thoroughly with DI water, drop-coated with the acrylic ation of small and uniform size of AcMPs was able to hold polymer microspheres at 3 mg/mL, and allowed to air-dry a large DNA-loading capacity and enhancing the sensitiv- at ambient conditions, followed by drop-casting with 5 mg/ ity and detection limit of the electrochemical arowana mL of colloidal AuNPs. The electrochemical characteristic DNA biosensor. of carbon SPE before and after modification with AcMPs and AuNPs was examined with CV method. Figure 1 por- Methods trays the method, which is composed of 3-step fabrication Apparatus and Electrodes of electrochemical DNA biosensor and 1-step arowana All the electrochemical measurements were performed cDNA detection. About 10 μLofcolloidal AuNPs(1mg/ with DPV using Autolab PGSTAT 12 potentiostat/galva- 300 μL) was firstly deposited onto a carbon SPE and air nostat (Metrohm) at 0.02 V step potential within the po- dried at 25 °C. As the AcMPs (1 mg) was readily suspended tential window of −1.0 V to −0.1 V. SPE from Scrint in ethanol (100 μL) to form a stable dispersion, 10 μLof Technology Co Malaysia modified with AcMPs and AcMP suspension was drop-coated onto the AuNP- AuNPs was used as the working electrode. A rod-shaped modified SPE. The AcMP-AuNP-modified carbon SPE was platinum (Pt) electrode and an Ag/AgCl electrode filled then dipped in 300 μLof5 μM arowana DNA probe solu- with 3.0 M of KCl internal solution were used as auxiliary tion for 6 h for DNA immobilisation process to take place and reference electrodes, respectively. Elma S30H sonica- and washed carefully with K-phosphate buffer (0.05 M, pH tor bath was used to prepare homogeneous solutions. 7.0) for three times to remove the unbound capture probe. The immobilised DNA probe was later immersed in Chemicals 300 μL of target DNA solution containing 2 M of NaCl and 2–2-Dimethoxy-2-phenylacetophenone (DMPP) was pur- 1 mM of AQMS to allow DNA hybridisation and intercal- chased from Fluka. 1,6-Hexanediol diacrylate (HDDA), n- ation reactions to occur within an hour, followed by se- butyl acrylate (nBA), and Au (III) chloride trihydrate were quentially rinsing with Milli-Q water and Na-phosphate supplied by Sigma-Aldrich. The colloidal AuNPs was syn- buffer (0.05 M, pH 7.0) for the removal of non-hybridised thesised according to the method reported by Grabar et DNA fragments and a specific binding of AQMS electro- al. (1995). Sodium dodecyl sulphate (SDS) and NaCl were chemical label. All the DPV measurements were performed obtained from Systerm. NAS and anthraquinone-2- in 4.5mL of0.05M of K-phosphatebufferatpH7.0 and sulfonic acid monohydrate sodium salt (AQMS) were pro- room temperature. cured from Acros. Milli-Q water (18 mΩ) was used to prepare all the chemical and biological solutions. Stock so- Optimization of Electrochemical Arowana DNA Biosensor lution of DNA probe was diluted with 0.05 M of K- The DNA electrodes modified with the respective AcMP, phosphate buffer (pH 7.0) while complementary DNA AuNP, and AcMP-AuNP composite were used in the (cDNA) and non-complementary (ncDNA) solutions were cDNA (5 μM) and ncDNA (5 μM) testing with DPV prepared with 0.05 M of Na-phosphate buffer at pH 7.0 containing 1.0 mM of AQMS. The K-phosphate buffer fa- cilitates maximum DNA probe immobilisation on the succinimide-functionalised acrylic material, whereas the Na-phosphate buffer provides an optimum condition for DNA hybridisation reaction [21, 22]. Synthesis of Acrylic Microsphere AcMPs were prepared according to the methods described previously with slight modification [22]. Briefly, a mixture of 450 μLofHDDA, 0.01 gofSDS, 0.1 gofDMPP, 7mLof nBA monomer, and 6 mg of NAS was dissolved into 15 mL of Milli-Q water and sonicated at room temperature (25 ° C) for 10 min. After that, the emulsion solution was photo- cured with UV light for 600 s under a continuous flow of N gas. The resulting poly(nBA-NAS) microspheres were then collected by centrifugation at 4000 rpm for 30 min Fig. 1 The fabrication procedure of electrochemical arowana DNA followed by washing in K-phosphate buffer (0.05 M, pH biosensor based on AcMP-AuNP-modified electrode 7.0) for three times and left to dry at ambient temperature. Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 4 of 10 electroanalytical method in the presence of 1 mM of AQMS and 2 M of NaCl at the scan rate of 0.5 V/s ver- sus Ag/AgCl reference electrode. DNA probe immobil- isation duration was determined by separately soaking nine units of AcMP-AuNP-modified SPEs in 300 μLof 5 μM arowana DNA probe solution for 1, 2, 3, 5, 6, 7, 8, 12, and 18 h, before reaction with 5 μM of cDNA in DNA hybridisation buffer (0.05 M of Na-phosphate buf- fer at pH 7.0) containing 1 mM of antraquinone redox intercalator and 2 M of NaCl. DNA hybridisation time was investigated by immersing the DNA electrode in 300 μLof 5 μM cDNA solution in the presence of 2 M of NaCl and 1 mM of AQMS for 10–100 min. The effect of temperature on the DNA hybridisation duration was done by measuring the arowana DNA biosensor re- sponse at 4, 25, 40, and 50 °C for an experimental period of 5–90 min in the measuring buffer using DPV tech- nique. For pH effect study, the arowana DNA biosensor was dipped in 5 μM of cDNA solution prepared from 0.05 M of Na-phosphate buffer conditioned with 2 M of NaCl and 1 mM of AQMS between pH 5.5 and pH 8.0 followed by DPV measurement. The effect of various 2+ + + 3+ positively charged ions (i.e. Ca ,Na ,K , and Fe ions) Fig. 2 SEM image of acrylic polymer microspheres on the electrochemical arowana DNA biosensor re- sponse was carried out by adding CaCl , NaCl, KCl, and FeCl into 0.05 M of Na-phosphate buffer (pH 7.0) prior DPV responses obtained were compared with the baseline to DNA hybridisation reaction and DPV measurement. current obtained without the presence of arowana DNA. Ionic strength of the hybridisation buffer was optimised A t test was applied to determine significant difference be- by varying the Na-phosphate buffer and NaCl concentra- tween the DNA biosensor response and baseline current tions from 0.002–0.1000 M to 1.52–5.50 M, respectively. at 4 degrees of freedom and 95% confidence level. The The linear calibration curve of the arowana DNA bio- DNA biosensor response obtained at significantly higher sensor was then established through quantitative meas- than the baseline current indicated a male arowana fish urement of a series of cDNA concentrations from was detected and vice versa. −18 −2 1.0 × 10 to 2.0 × 10 μM via DPV method. All the experiments were performed in triplicate. Results and Discussion The as-synthesised AcMPs were observed (Fig. 2) under DNA Extraction and Arowana DNA Analysis scanning electron microscope (SEM, LEO 1450VP). The A total of 15 arowana fish tissue samples were kindly pro- vided by Fisheries Research Institute (FRI), Department of Fisheries Malaysia. All the fish tissue samples were stored in 70% ethanol in a chiller at 4 °C and dispatched to the laboratory. The fish tissue samples were washed with Milli-Q water and cut into small pieces and dried at ambi- ent conditions before kept in the freezer at −20 °C. Aro- wana DNA from each tissue sample (35–40 mg each) was then separately extracted using QIAquick PCR Purifica- tion kit (Manchester, UK) according to the manufacturer’s protocol and stored at −20 °C when not in use. PCR amp- lification of genomic DNA fragment was then performed using Bio-Rad PCR thermal cycler (PTC-100, Hercules, USA). The DNA fragments of PCR product were then separated with 1.5% agarose gel electrophoresis. The aro- Fig. 3 Size distribution of acrylic micropsheres prepared wana DNA extracts were also analysed by the electro- from photopolymerisation chemical DNA biosensor to determine the gender. The Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 5 of 10 correlation coefficient (R ) of 0.996 within the range of 50–300 mV/s as shown by Eq. 2 and Fig. 5a. 1=2– ip ¼ 1:463v 2:451 ð2Þ This indicates that the reaction at the surface of the modi- fied electrode was a diffusion controlled reaction [22–25]. Furthermore, based on Fig. 5b, when the log value of oxidation current was plotted against the log value of scan rate, a linear line was obtained with a slope of 0.65, which was close to the theoretical value of 0.50 for diffusion-controlled process. Therefore, the study has demonstrated that the reaction at the surface of the modified SPE is mostly diffusion controlled. For the ideal case of a fast, reversible, and one-electron transfer process, ΔEp = 0.059 V at 298 K. However, the peak potential shifts that increased with the scan rate Fig. 4 Cyclic voltammograms of 1.0 mM K Fe(CN) in 0.05 M 3 6 Na-phosphate buffer of pH 7.0 with different scan rates (0.05, demonstrated larger peak potential separations of more 0.10, 0.15, 0.20, 0.25, and 0.30 V/s) for a modified carbon SPE than 0.059 V (Fig 4). This implies that the electron trans- containing AcMP-AuNP material at the electrode surface fer process at the electrode surface is slow [22, 25, 26], probably due to the resistance created by the presence of size distribution of acrylic miscropsheres prepared from AcMP material covering the electrode surface. photopolymersation is illustrated in Fig. 3. Figure 6 shows the DPV response of arowana DNA bio- The effect of the different scan rates of the carbon SPE sensor based on AcMP, AuNP, and AcMP-AuNP-modified containing AcMPs-AuNPs in the presence of K Fe(CN) carbon SPEs. The significant DPV current difference ob- 3 6 showed that the oxidation and reduction peak currents in- served between experiment (a) and (c) reveals that the aro- creased with the increasing of the scan rate from 0.05 to wana DNA probes were successfully grafted onto the 0.30 V/s (Fig. 4). Thus, the electron transfer process at the AcMPs via strong covalent bonds between succinimide electrode surface is expected to be reversible [22–25]. functional group of AcMP and amine functional group of Based on the Randles–Sevcik equation, the aminated DNA probe, and the immobilised arowana DNA probe was selective only to its cDNA [19, 20]. The AuNPs played a role to assist the electron conductivity 1=2 ip ¼ 0:4463 nFACðÞ nFvD=RT ð1Þ from the intercalated AQMS to the fabricated electrode surface. Without the inclusion of AuNPs in the composite a good linearity was found between the redox peak material (f), only gold nanoparticles (e), and the gold nano- current and the square root of the scan rate with a particles and AcMP composite (d), only very little current 1/2 Fig. 5 Plot of the oxidation peak currents (ip/μA) versus square root of scan rate ((mV/s) )(a) and plot of log of oxidation peak currents (ip/μA) versus log of scan rates (log (mV/s)) (b) Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 6 of 10 was required to promote larger amount of DNA probes to be attached on the AcMP-AuNP-modified electrode. At a further prolonging of DNA probe immobilisation time, no noticeable change in the DNA biosensor response was perceived as the binding sites of immobilised AcMPs have fully bound with DNA probes. The arowana DNA biosen- sor response is also dependent on the DNA hybridisation time. The biosensor response profile illustrated in Fig. 7b shows an increasing DPV current response trend with DNA hybridisation duration from 10 to 60 min, after which the current response becomes almost plateau. At this stage, the immobilised DNA probes on the electrode have entirely hybridised with cDNA [29]. It is also noticed that the DNA hybridisation time of the fabricated arowana DNA biosensor was temperature dependent, and as a great advantage, we obtained a max- Fig. 6 The DPV signal of AcMP-AuNP-based DNA electrode upon imum current response at room temperature within hybridisation with cDNA (a) and non-complementary DNA (b), the 30 min (Fig. 8). At low temperature, i.e. 4 °C, a long time DPV response of the AcMPs (f) and AuNP-modified SPE (e), and was required for a complete DNA hybridisation reaction AcMP-AuNP composite modified SPE as well as the response of DNA biosensor based on AcMP-AuNP composite modified probe because the cold temperature slowed down the DNA hy- DNA SPE (c) before reaction with cDNA in the presence of 1 mM bridisation reaction rate. A faster DNA hybridisation AQMS at the scan rate of 0.5 V/s versus Ag/AgCl reference electrode time could be achieved at a temperature above 25 °C as- cribed to the higher DNA hybridisation reaction rate oc- response can be observed. The low DPV currents acquired curred between immobilised DNA probe and cDNA to in experiment (b) was due to no DNA hybridisation reac- form the duplex DNA at high temperatures. However, tion occurred with ncDNA, which also indicates no specific high temperature could permanently deform the double- absorptions of AQMS redox indicator on the electrode sur- helical structure of DNA, and regeneration of the DNA face [27, 28]. molecule is not possible even after the readjustment of For DNA probe immobilisation duration, Fig. 7a ex- the temperature to the optimal value [28, 30]. hibits the DNA biosensor response slowly increased over As part of the arowana DNA biosensor response opti- the first 1–3 h of DNA probe immobilisation time and the misation, the effect of solution pH on the DNA hybridisa- abrupt increase in the DNA biosensor response can be tion reaction was investigated. The DNA biosensor seen between 3 and 6 h of DNA probe immobilisation showed negligible current change between pH 5.5 and pH duration. This was because a longer immobilisation time 6.5 due to the protonation of phosphodiester backbone of Fig. 7 Effects of DNA probe immobilisation time (a) and DNA hybridization time (b) on the arowana DNA biosensor response using 5 μM DNA probe and cDNA in the presence of 1 mM AQMS at 2 M ionic strength Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 7 of 10 The effect of valency of cations towards DNA hybridisa- tion reaction was performed using different cations of 4 oC 2+ + + 3+ salts, e.g. Ca ,Na ,K ,and Fe ions in the DNA hybrid- isation buffer. The positively charged ions could interact 25 oC electrostatically with the negatively charged phospho- diester chain of DNA molecule to overcome the steric 40 oC hindrance and electrostatic repulsion between the immo- bilised DNA probe and target DNA, thereby facilitates the 50 oC DNA hybridisation process [34]. Figure 10 demonstrates that the DNA hybridisation reaction was favourable in the + + 3+ 2 presence of cations in the order of Na >K >Fe >Ca + 2+ 3+ . The presence of Ca and Fe ions were noticed to cause a remarkable decrement in the arowana DNA bio- 0 20 40 60 80 100 + + sensor current response compared to Na and K ions. Times (min) These phenomena were attributed to the formation of Fig. 8 Effect of temperature on the DNA hybridisation time of arowana sparingly soluble calcium phosphate and ferrum (III) DNA biosensor. The DPV response was measured in 0.05 M K-phosphate buffer (pH 7.0) at 4, 25, 40, and 50 °C for an experimental period phosphate salts in the DNA hybridisation buffer [22], of 5–90 min which reduced the ionic content of the solution and caused a high electrostatic repulsion between the DNA molecules. As a result, the DNA hybridisation rate was de- DNA, which reduced the solubility of DNA molecules in clined and led to a poor biosensor performance. The high- aqueous environment (Fig. 9). Further increase in pH of est DNA biosensor response was obtained when Na ions the DNA hybridisation medium, the arowana DNA bio- were added to the DNA hybridisation phosphate buffer sensor response increased abruptly at pH 7.0, after which because of their small size and strong affinity towards the a sharp decline in DPV current was discernible as the pH DNA phosphodieter bond. environment changed to basic condition due to the The concentration of NaCl and Na-phosphate buffer irreversible denaturation of DNA in the higher pH range (pH 7.0) must also be optimised to provide an optimal [23, 24, 31–33]. Since maximum DPV response was ac- ionic strength for hybridisation buffer. Figure 11b indi- quired at a neutral pH, the next electrochemical evaluation cates that ionic strength of below and above 2 M could of arowana DNA biosensor response was maintained at pH not overcome the high electrostatic repulsion between 7.0 using 0.05 M of Na-phosphate buffer. DNA strands. About 0.05 M of Na-phosphate buffer (Fig. 11a) and 2 M of NaCl were found to provide the optimum Fig. 9 The DPV response of arowana DNA biosensor based on AcMP- 2+ + + 3+ AuNP composite modified carbon SPE between pH 5.5 and pH 8.0. The Fig. 10 The effect of Ca ,Na ,K , and Fe ions in the DNA DPV measurement was conducted in 0.05 M K-phosphate buffer (pH 7.0) hybridisation buffer (0.05 M Na-phosphate buffer at pH 7.0) on the at 25 °C and scan rate of 0.5 V/s versus Ag/AgCl reference electrode DPV response of arowana DNA biosensor i / µA Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 8 of 10 ionic strength for the assay of arowana target DNA with Determination of Arowana Fish Gender with DNA maximum biosensor performance. Optimum hybridisa- Biosensor tion buffer conditions in terms of pH, buffer capacity, and The developed electrochemical DNA biosensor has been ionic strength would allow DNA hybridisation reaction to validated with the standard PCR-based method to deter- occur at the most minimum steric hindrance [30]. mine the gender of Asian arowana fish. With the results The optimised DNA biosensor was then used for the de- tabulated in Table 2, both methods provided the same re- tection of a series of arowana cDNA concentrations be- sult for the gender determination of arowana fish. This in- −12 −2 tween 1.0 × 10 and 1.0 × 10 μM. The DNA biosensor dicates that the proposed DNA biosensor can be used for −18 showed a wide linear response range from 1.0 × 10 to accurate determination of arowana gender in a simple and −8 2 1.0 × 10 M(R = 0.99). The limit of detection (LOD) ob- fast way. −18 tained at 1.0 × 10 M was calculated based on three times the standard deviation of biosensor response at the re- Conclusions sponse curve approximating LOD divided by the linear cali- The electrochemical DNA biosensor developed in this study bration slope. The homogeneous AcMP particles size demonstrated good sensitivity, wide linear response ranges, within micrometre range exhibited a significant influence and low detection limit in the determination of arowana tar- on the DNA biosensor sensitivity and reproducibility get DNA. In addition, the DNA biosensor showed a good (RSD =5.6%).The largebinding surfaceareaof the immo- response towards arowana cDNA, which implies that the bilised NAS-functionalised AcMPs permitted a large num- electrochemical DNA biosensor could be used to success- ber of DNA molecules to bind covalently to the electrode fully detect the arowana DNA segments. The developed surface, thereby increasing the DNA biosensor analytical arowana DNA biosensor can be further redesigned into a performance with respect to dynamic linear range and de- point-of-use device prototype that offers a great potential tection limit of the arowana DNA biosensor (Fig. 12). Fig. 11 The arowana DNA biosensor response trends as the a Na- phosphate buffer concentration and b ionic strength of the hybrid- Fig. 12 The arowana DNA biosensor response curve (a) and linear isation buffer varied from 0.002–0.100 M and calibration range (b) and the DPV voltammogram (c) obtained using −18 −2 1.52–5.50 M, respectively 1.0 × 10 to 1.0 × 10 μM cDNA at pH 7.0 Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 9 of 10 Table 2 A comparison between DNA biosensor and PCR method in the gender identification of arowana fish using fish tissue samples No Sample DNA biosensor method PCR method Current (μA) RSD Baseline ± SD t test Gender 1 227 1.479 ± 0.138 9.360 1.885 ± 0.10 5.042** FF 2 231 2.315 ± 0.149 6.453 1.885 ± 0.10 7.184** MM 3 232 2.627 ± 0.185 7.053 1.885 ± 0.10 7.219** MM 4 233 1.829 ± 0.158 8.643 1.885 ± 0.10 1.117 F F 5 236 2.021 ± 0.169 8.372 1.885 ± 0.10 1.387 F F 6 417 2.947 ± 0.215 7.291 1.956 ± 0.06 10.412** MM 7 437 2.779 ± 0.089 3.217 1.956 ± 0.06 22.126** MM 8 450 1.964 ± 0.122 6.215 1.956 ± 0.06 0.093 F F 9 530 2.500 ± 0.232 9.264 1.956 ± 0.06 4.542** MM 10 531 2.581 ± 0.195 7.556 1.956 ± 0.06 6.544** MM 11 537 2.001 ± 0.189 9.441 1.993 ± 0.12 0.124 F F 12 521 1.672 ± 0.043 2.600 1.993 ± 0.12 6.599** FF 13 524 2.774 ± 0.102 3.678 1.993 ± 0.12 10.775** MM 14 525 1.359 ± 0.075 5.512 1.993 ± 0.12 7.377** FF 15 526 2.953 ± 0.169 5.731 1.993 ± 0.12 10.857** MM M male, F female Y X ** –DPV current significantly higher than the baseline current (obtained from PBS buffer alone) indicates male fish; ** –DPV current significantly lower than the baseline current indicates female fish, critical value t = 2.78 (p = 0.05, 95%) for the application in the fish culture for early identification Received: 4 April 2017 Accepted: 27 July 2017 of arowana gender and colour, which is economically ad- vantageous in fishery and aquaculture sectors. 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International Journal of Biology 1(2):28 Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 7. Bonde N (1979) Palaeoenvironment in the “North Sea” as indicated by the fish bearing Mo− Clay deposit (Paleocene/Eocene), Denmark. Mededelingen Author details van de Werkgroep voor Tertiaire en Kwartaire Geologie 16(1):3–16 Department of General Educational Development (GED), Faculty of Science 8. Mu XD, Song HM, Wang XJ, Yang YX, Luo D, Gu DE, Luo JR, Hu YC (2012) & Information Technology, Daffodil International University, Dhanmondi, Genetic variability of the Asian arowana, Scleropages formosus, based on Dhaka 1207, Bangladesh. School of Chemical Sciences and Food mitochondrial DNA genes. Biochem Syst Ecol 44:141–148 Technology, Faculty of Science and Technology, Universiti Kebangsaan 9. Alfred E (1964) The fresh-water food fishes of Malaya. I. 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A Highly Sensitive Electrochemical DNA Biosensor from Acrylic-Gold Nano-composite for the Determination of Arowana Fish Gender

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Materials Science; Nanotechnology; Nanotechnology and Microengineering; Nanoscale Science and Technology; Nanochemistry; Molecular Medicine
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Abstract

The present research describes a simple method for the identification of the gender of arowana fish (Scleropages formosus). The DNA biosensor was able to detect specific DNA sequence at extremely low level down to atto M regimes. An electrochemical DNA biosensor based on acrylic microsphere-gold nanoparticle (AcMP-AuNP) hybrid composite was fabricated. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesised with a facile and well-established one-step photopolymerization procedure and physically adsorbed on the AuNPs at the surface of a carbon screen printed electrode (SPE). The DNA biosensor was constructed simply by grafting an aminated DNA probe on the succinimide functionalised AcMPs via a strong covalent attachment. DNA hybridisation response was determined by differential pulse voltammetry (DPV) technique using anthraquinone monosulphonic −18 acid redox probe as an electroactive oligonucleotide label (Table 1). A low detection limit at 1.0 × 10 M with a −18 −8 2 wide linear calibration range of 1.0 × 10 to 1.0 × 10 M(R = 0.99) can be achieved by the proposed DNA biosensor under optimal conditions. Electrochemical detection of arowana DNA can be completed within 1 hour. Due to its small size and light weight, the developed DNA biosensor holds high promise for the development of functional kit for fish culture usage. Keywords: DNA biosensor, Electrochemical biosensor, Acrylic microspheres, DNA hybridization, Photopolymerization, Arowana DNA Background good luck and happiness, along with many other cultures Asiatic arowana (Scleropages formoss), a freshwater fish, [1] [6]. Generally, the arowana is around 7 kg weight and 1 m is widely distributed over the countryside of Southeast Asia long in their mature age [9]. This ornamental fish possesses region such as Malaysia, Singapore, Thailand, Indonesia, attractive colours and morphology and can be identified by Cambodia, Vietnam, Laos, Myanmar and the Philippines. its distinctive physical features, such as comparatively long In addition, the arowana fish is also found in Australia and in body size, a large pectoral fin, and the dorsal and anal New Guinea [1–4]. It is popularly known as dragonfish, fins are positioned far back on the body. There are three Asia bonytongue, kelisa, or baju-rantai [5, 6]. It is still sur- main colour varieties, i.e. golden, red, and green of closely viving as a primitive fish species from the Jurassic era [7, 8]. related freshwater fish within the Asian arowana species. The Chinese and Asian people considered it as a symbol of There are also several other distinct species derived from different parts of the Southeast Asia and are regional to many river systems [8]. * Correspondence: mahbub_iuchem@yahoo.com Department of General Educational Development (GED), Faculty of Science Due to its high popularity and great demand in ornamen- & Information Technology, Daffodil International University, Dhanmondi, tal purposes, Asian arowana has been fiercely hunted for Dhaka 1207, Bangladesh profits [6], and results in a rapid decline of its population. School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Considering its high demand in ornamental industry, the Darul Ehsan, Malaysia over-exploitation of natural populations, and the rarity of Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 2 of 10 Table 1 Sequences of oligonucleotides utilised in the present the attempt to identify the gender and colour at their early investigation age. Traditional method based on body size and mouth DNA Base sequences cavity estimations can only be made at around 3 months of age of baby arowana for gender and colour identifica- DNA Probe 5'-AAT TCA AGG GAA CTG ATG ACT CTA (AmC7) tions [17]. However, this conventional visual examination cDNA 5'-TAG AGT CAT CAG TTC CCT TGA ATT method is time-consuming and often provides inaccurate ncDNA 5'-CGA GCG ACG TGA GCT TAG CTG CGC result. On the other hand, the widely used standard methods based on DNA sequencing, i.e., polymerase chain natural habitats due to changes in the living environment, reaction (PCR) and gel electrophoresis are labour-, time-, Asian arowana has been classified as an endangered species and resource-demanding. An alternative algorithm of in- threatened with extinction since 1980 by the Convention ventive problem solving (ARIZ) method was previously on International Trade in Endangered Species of Wild employed for the detection of arowana gender detection Faunaand Flora(CITES) andhas recently listed as endan- [18]. ARIZ is an alternative tool for gender detection, con- gered by the 2006 IUCN Red List [1, 3, 8, 10, 11]. However, taining nine different parts and a total of 40 complex the commercial trading of this endangered species is pro- steps. It requires a very long time to learn and practice hibited under CITES except in certain countries, e.g., and demands highly experienced personnel to operate. Indonesia, Singapore, and Malaysia. [2, 3, 12]. There are a For example, the application of ARIZ in various engineer- number of CITES registered cultivators in Asia actively car- ing systems has been employed, but most of the cases did rying out farming and trading of arowana fish [2, 12]. This not cover all the requirements and processes of ARIZ. Asian freshwater fish consists of geographically isolated In this research, acrylic polymer microspheres strains, and it is the only member of the species with differ- modified with succinimide functional groups via N- ent colour varieties that is based on different geographic acryloxysuccinimide (NAS) moieties was used as the distributions throughout the rivers of Southeast Asia. The matrix for DNA probe immobilisation. As previously re- species distribution is now far more widespread, which ex- ported by Chen and Chiu 2000 and Chaix et al. 2003 tends to the Nile River of Africa, the Amazon River of [19, 20], the succinimide functional group can react with South America, Australia, and New Guinea [1, 4, 8]. amine functional groups to form a covalent bond. The Among the different colours of Asiatic arowana, red and incorporation of NAS functionality into acrylic micro- golden arowana fishes are the most expensive and popular spheres for DNA microbiosensor application provides ornamental pets in the hatchery industry compared to advantages of a simple preparation method where the black, green, silver, and others colour varieties [1, 5, 10, 13]. spheres can be synthesised and functionalised via a one- The egg thievery phenomenon of Asiatic arowana is atyp- step procedure using photopolymerisation in a short ical compared to other fish species. In general, arowana duration (several minutes). In addition, the microspheres fishes get mature at the age of 3–4 years, and they lay only have the advantage of small size and provide a large sur- afew eggs (30–100) [14, 15] of extra-large size (around face area for DNA probe immobilisation, thus reducing 1 cm in diameter) [16]. Interestingly, the fertilised eggs and the barrier to diffusion for reactants and products. This larvae are then protected and grown up in the mouth of enables the improvement in the biosensor performance male arowana fishes, and they show high parental care. To in terms of shorter response times and wider linear re- identify the gender based on visual observation of the baby sponse range, which will be demonstrated in the work arowana is difficult because there is no distinctive pheno- reported here. typic organ of sexual dimorphism [14, 15]. Only one of the In this study, an electrochemical DNA biosensor parents (presume to be the male) can be identified as the method, which is highly sensitive, simple, easy-to- offspring are harvested from his mouth. The other parent fabricate, and low cost, is proposed for juvenile arowana cannot be identified from among a number of potential fish gender determination with high accuracy. The DNA parents [16]. biosensor was built from a carbon screen printed elec- Usually, the hobbyists keep the baby arowana fish for trode (SPE) modified with colloidal gold nanoparticles their ornamental purposes in the aquarium as well as for (AuNPs) and polyacrylate microspheres functionalised cultivation in the fish farm. However, all types of juvenile with NAS functional group. The AuNPs were immobilised arowana fishes are sold at the same price, because of the onto the carbon SPE surface via electrostatistic interaction lack of assistive technology for gender and colour variety and played an important role in enhancing the electrode differentiation. Until the present time, there is no estab- conductivity and facilitating the electron transfer, while lished method published to identify the gender and colour the acrylic microspheres (AcMPs) were directly deposited of arowana fishes at their juvenile stage. Instead, hundreds onto the AuNP-modified SPE via physical adsorption. of studies have been carried out using DNA analysis based Aminated DNA probe of arowana was then covalently at- on genetic structure and biography of arowana fishes in tached to the immobilised AcMP-AuNP composite at the Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 3 of 10 exposed succinimide group of AcMPs. Probe-target hy- Fabrication of DNA Biosensor Using Acrylic Microspheres bridisation was detected with anthraquinone redox label Prior to surface modification, the carbon SPE was rinsed via differential pulse voltammetry (DPV). The incorpor- thoroughly with DI water, drop-coated with the acrylic ation of small and uniform size of AcMPs was able to hold polymer microspheres at 3 mg/mL, and allowed to air-dry a large DNA-loading capacity and enhancing the sensitiv- at ambient conditions, followed by drop-casting with 5 mg/ ity and detection limit of the electrochemical arowana mL of colloidal AuNPs. The electrochemical characteristic DNA biosensor. of carbon SPE before and after modification with AcMPs and AuNPs was examined with CV method. Figure 1 por- Methods trays the method, which is composed of 3-step fabrication Apparatus and Electrodes of electrochemical DNA biosensor and 1-step arowana All the electrochemical measurements were performed cDNA detection. About 10 μLofcolloidal AuNPs(1mg/ with DPV using Autolab PGSTAT 12 potentiostat/galva- 300 μL) was firstly deposited onto a carbon SPE and air nostat (Metrohm) at 0.02 V step potential within the po- dried at 25 °C. As the AcMPs (1 mg) was readily suspended tential window of −1.0 V to −0.1 V. SPE from Scrint in ethanol (100 μL) to form a stable dispersion, 10 μLof Technology Co Malaysia modified with AcMPs and AcMP suspension was drop-coated onto the AuNP- AuNPs was used as the working electrode. A rod-shaped modified SPE. The AcMP-AuNP-modified carbon SPE was platinum (Pt) electrode and an Ag/AgCl electrode filled then dipped in 300 μLof5 μM arowana DNA probe solu- with 3.0 M of KCl internal solution were used as auxiliary tion for 6 h for DNA immobilisation process to take place and reference electrodes, respectively. Elma S30H sonica- and washed carefully with K-phosphate buffer (0.05 M, pH tor bath was used to prepare homogeneous solutions. 7.0) for three times to remove the unbound capture probe. The immobilised DNA probe was later immersed in Chemicals 300 μL of target DNA solution containing 2 M of NaCl and 2–2-Dimethoxy-2-phenylacetophenone (DMPP) was pur- 1 mM of AQMS to allow DNA hybridisation and intercal- chased from Fluka. 1,6-Hexanediol diacrylate (HDDA), n- ation reactions to occur within an hour, followed by se- butyl acrylate (nBA), and Au (III) chloride trihydrate were quentially rinsing with Milli-Q water and Na-phosphate supplied by Sigma-Aldrich. The colloidal AuNPs was syn- buffer (0.05 M, pH 7.0) for the removal of non-hybridised thesised according to the method reported by Grabar et DNA fragments and a specific binding of AQMS electro- al. (1995). Sodium dodecyl sulphate (SDS) and NaCl were chemical label. All the DPV measurements were performed obtained from Systerm. NAS and anthraquinone-2- in 4.5mL of0.05M of K-phosphatebufferatpH7.0 and sulfonic acid monohydrate sodium salt (AQMS) were pro- room temperature. cured from Acros. Milli-Q water (18 mΩ) was used to prepare all the chemical and biological solutions. Stock so- Optimization of Electrochemical Arowana DNA Biosensor lution of DNA probe was diluted with 0.05 M of K- The DNA electrodes modified with the respective AcMP, phosphate buffer (pH 7.0) while complementary DNA AuNP, and AcMP-AuNP composite were used in the (cDNA) and non-complementary (ncDNA) solutions were cDNA (5 μM) and ncDNA (5 μM) testing with DPV prepared with 0.05 M of Na-phosphate buffer at pH 7.0 containing 1.0 mM of AQMS. The K-phosphate buffer fa- cilitates maximum DNA probe immobilisation on the succinimide-functionalised acrylic material, whereas the Na-phosphate buffer provides an optimum condition for DNA hybridisation reaction [21, 22]. Synthesis of Acrylic Microsphere AcMPs were prepared according to the methods described previously with slight modification [22]. Briefly, a mixture of 450 μLofHDDA, 0.01 gofSDS, 0.1 gofDMPP, 7mLof nBA monomer, and 6 mg of NAS was dissolved into 15 mL of Milli-Q water and sonicated at room temperature (25 ° C) for 10 min. After that, the emulsion solution was photo- cured with UV light for 600 s under a continuous flow of N gas. The resulting poly(nBA-NAS) microspheres were then collected by centrifugation at 4000 rpm for 30 min Fig. 1 The fabrication procedure of electrochemical arowana DNA followed by washing in K-phosphate buffer (0.05 M, pH biosensor based on AcMP-AuNP-modified electrode 7.0) for three times and left to dry at ambient temperature. Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 4 of 10 electroanalytical method in the presence of 1 mM of AQMS and 2 M of NaCl at the scan rate of 0.5 V/s ver- sus Ag/AgCl reference electrode. DNA probe immobil- isation duration was determined by separately soaking nine units of AcMP-AuNP-modified SPEs in 300 μLof 5 μM arowana DNA probe solution for 1, 2, 3, 5, 6, 7, 8, 12, and 18 h, before reaction with 5 μM of cDNA in DNA hybridisation buffer (0.05 M of Na-phosphate buf- fer at pH 7.0) containing 1 mM of antraquinone redox intercalator and 2 M of NaCl. DNA hybridisation time was investigated by immersing the DNA electrode in 300 μLof 5 μM cDNA solution in the presence of 2 M of NaCl and 1 mM of AQMS for 10–100 min. The effect of temperature on the DNA hybridisation duration was done by measuring the arowana DNA biosensor re- sponse at 4, 25, 40, and 50 °C for an experimental period of 5–90 min in the measuring buffer using DPV tech- nique. For pH effect study, the arowana DNA biosensor was dipped in 5 μM of cDNA solution prepared from 0.05 M of Na-phosphate buffer conditioned with 2 M of NaCl and 1 mM of AQMS between pH 5.5 and pH 8.0 followed by DPV measurement. The effect of various 2+ + + 3+ positively charged ions (i.e. Ca ,Na ,K , and Fe ions) Fig. 2 SEM image of acrylic polymer microspheres on the electrochemical arowana DNA biosensor re- sponse was carried out by adding CaCl , NaCl, KCl, and FeCl into 0.05 M of Na-phosphate buffer (pH 7.0) prior DPV responses obtained were compared with the baseline to DNA hybridisation reaction and DPV measurement. current obtained without the presence of arowana DNA. Ionic strength of the hybridisation buffer was optimised A t test was applied to determine significant difference be- by varying the Na-phosphate buffer and NaCl concentra- tween the DNA biosensor response and baseline current tions from 0.002–0.1000 M to 1.52–5.50 M, respectively. at 4 degrees of freedom and 95% confidence level. The The linear calibration curve of the arowana DNA bio- DNA biosensor response obtained at significantly higher sensor was then established through quantitative meas- than the baseline current indicated a male arowana fish urement of a series of cDNA concentrations from was detected and vice versa. −18 −2 1.0 × 10 to 2.0 × 10 μM via DPV method. All the experiments were performed in triplicate. Results and Discussion The as-synthesised AcMPs were observed (Fig. 2) under DNA Extraction and Arowana DNA Analysis scanning electron microscope (SEM, LEO 1450VP). The A total of 15 arowana fish tissue samples were kindly pro- vided by Fisheries Research Institute (FRI), Department of Fisheries Malaysia. All the fish tissue samples were stored in 70% ethanol in a chiller at 4 °C and dispatched to the laboratory. The fish tissue samples were washed with Milli-Q water and cut into small pieces and dried at ambi- ent conditions before kept in the freezer at −20 °C. Aro- wana DNA from each tissue sample (35–40 mg each) was then separately extracted using QIAquick PCR Purifica- tion kit (Manchester, UK) according to the manufacturer’s protocol and stored at −20 °C when not in use. PCR amp- lification of genomic DNA fragment was then performed using Bio-Rad PCR thermal cycler (PTC-100, Hercules, USA). The DNA fragments of PCR product were then separated with 1.5% agarose gel electrophoresis. The aro- Fig. 3 Size distribution of acrylic micropsheres prepared wana DNA extracts were also analysed by the electro- from photopolymerisation chemical DNA biosensor to determine the gender. The Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 5 of 10 correlation coefficient (R ) of 0.996 within the range of 50–300 mV/s as shown by Eq. 2 and Fig. 5a. 1=2– ip ¼ 1:463v 2:451 ð2Þ This indicates that the reaction at the surface of the modi- fied electrode was a diffusion controlled reaction [22–25]. Furthermore, based on Fig. 5b, when the log value of oxidation current was plotted against the log value of scan rate, a linear line was obtained with a slope of 0.65, which was close to the theoretical value of 0.50 for diffusion-controlled process. Therefore, the study has demonstrated that the reaction at the surface of the modified SPE is mostly diffusion controlled. For the ideal case of a fast, reversible, and one-electron transfer process, ΔEp = 0.059 V at 298 K. However, the peak potential shifts that increased with the scan rate Fig. 4 Cyclic voltammograms of 1.0 mM K Fe(CN) in 0.05 M 3 6 Na-phosphate buffer of pH 7.0 with different scan rates (0.05, demonstrated larger peak potential separations of more 0.10, 0.15, 0.20, 0.25, and 0.30 V/s) for a modified carbon SPE than 0.059 V (Fig 4). This implies that the electron trans- containing AcMP-AuNP material at the electrode surface fer process at the electrode surface is slow [22, 25, 26], probably due to the resistance created by the presence of size distribution of acrylic miscropsheres prepared from AcMP material covering the electrode surface. photopolymersation is illustrated in Fig. 3. Figure 6 shows the DPV response of arowana DNA bio- The effect of the different scan rates of the carbon SPE sensor based on AcMP, AuNP, and AcMP-AuNP-modified containing AcMPs-AuNPs in the presence of K Fe(CN) carbon SPEs. The significant DPV current difference ob- 3 6 showed that the oxidation and reduction peak currents in- served between experiment (a) and (c) reveals that the aro- creased with the increasing of the scan rate from 0.05 to wana DNA probes were successfully grafted onto the 0.30 V/s (Fig. 4). Thus, the electron transfer process at the AcMPs via strong covalent bonds between succinimide electrode surface is expected to be reversible [22–25]. functional group of AcMP and amine functional group of Based on the Randles–Sevcik equation, the aminated DNA probe, and the immobilised arowana DNA probe was selective only to its cDNA [19, 20]. The AuNPs played a role to assist the electron conductivity 1=2 ip ¼ 0:4463 nFACðÞ nFvD=RT ð1Þ from the intercalated AQMS to the fabricated electrode surface. Without the inclusion of AuNPs in the composite a good linearity was found between the redox peak material (f), only gold nanoparticles (e), and the gold nano- current and the square root of the scan rate with a particles and AcMP composite (d), only very little current 1/2 Fig. 5 Plot of the oxidation peak currents (ip/μA) versus square root of scan rate ((mV/s) )(a) and plot of log of oxidation peak currents (ip/μA) versus log of scan rates (log (mV/s)) (b) Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 6 of 10 was required to promote larger amount of DNA probes to be attached on the AcMP-AuNP-modified electrode. At a further prolonging of DNA probe immobilisation time, no noticeable change in the DNA biosensor response was perceived as the binding sites of immobilised AcMPs have fully bound with DNA probes. The arowana DNA biosen- sor response is also dependent on the DNA hybridisation time. The biosensor response profile illustrated in Fig. 7b shows an increasing DPV current response trend with DNA hybridisation duration from 10 to 60 min, after which the current response becomes almost plateau. At this stage, the immobilised DNA probes on the electrode have entirely hybridised with cDNA [29]. It is also noticed that the DNA hybridisation time of the fabricated arowana DNA biosensor was temperature dependent, and as a great advantage, we obtained a max- Fig. 6 The DPV signal of AcMP-AuNP-based DNA electrode upon imum current response at room temperature within hybridisation with cDNA (a) and non-complementary DNA (b), the 30 min (Fig. 8). At low temperature, i.e. 4 °C, a long time DPV response of the AcMPs (f) and AuNP-modified SPE (e), and was required for a complete DNA hybridisation reaction AcMP-AuNP composite modified SPE as well as the response of DNA biosensor based on AcMP-AuNP composite modified probe because the cold temperature slowed down the DNA hy- DNA SPE (c) before reaction with cDNA in the presence of 1 mM bridisation reaction rate. A faster DNA hybridisation AQMS at the scan rate of 0.5 V/s versus Ag/AgCl reference electrode time could be achieved at a temperature above 25 °C as- cribed to the higher DNA hybridisation reaction rate oc- response can be observed. The low DPV currents acquired curred between immobilised DNA probe and cDNA to in experiment (b) was due to no DNA hybridisation reac- form the duplex DNA at high temperatures. However, tion occurred with ncDNA, which also indicates no specific high temperature could permanently deform the double- absorptions of AQMS redox indicator on the electrode sur- helical structure of DNA, and regeneration of the DNA face [27, 28]. molecule is not possible even after the readjustment of For DNA probe immobilisation duration, Fig. 7a ex- the temperature to the optimal value [28, 30]. hibits the DNA biosensor response slowly increased over As part of the arowana DNA biosensor response opti- the first 1–3 h of DNA probe immobilisation time and the misation, the effect of solution pH on the DNA hybridisa- abrupt increase in the DNA biosensor response can be tion reaction was investigated. The DNA biosensor seen between 3 and 6 h of DNA probe immobilisation showed negligible current change between pH 5.5 and pH duration. This was because a longer immobilisation time 6.5 due to the protonation of phosphodiester backbone of Fig. 7 Effects of DNA probe immobilisation time (a) and DNA hybridization time (b) on the arowana DNA biosensor response using 5 μM DNA probe and cDNA in the presence of 1 mM AQMS at 2 M ionic strength Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 7 of 10 The effect of valency of cations towards DNA hybridisa- tion reaction was performed using different cations of 4 oC 2+ + + 3+ salts, e.g. Ca ,Na ,K ,and Fe ions in the DNA hybrid- isation buffer. The positively charged ions could interact 25 oC electrostatically with the negatively charged phospho- diester chain of DNA molecule to overcome the steric 40 oC hindrance and electrostatic repulsion between the immo- bilised DNA probe and target DNA, thereby facilitates the 50 oC DNA hybridisation process [34]. Figure 10 demonstrates that the DNA hybridisation reaction was favourable in the + + 3+ 2 presence of cations in the order of Na >K >Fe >Ca + 2+ 3+ . The presence of Ca and Fe ions were noticed to cause a remarkable decrement in the arowana DNA bio- 0 20 40 60 80 100 + + sensor current response compared to Na and K ions. Times (min) These phenomena were attributed to the formation of Fig. 8 Effect of temperature on the DNA hybridisation time of arowana sparingly soluble calcium phosphate and ferrum (III) DNA biosensor. The DPV response was measured in 0.05 M K-phosphate buffer (pH 7.0) at 4, 25, 40, and 50 °C for an experimental period phosphate salts in the DNA hybridisation buffer [22], of 5–90 min which reduced the ionic content of the solution and caused a high electrostatic repulsion between the DNA molecules. As a result, the DNA hybridisation rate was de- DNA, which reduced the solubility of DNA molecules in clined and led to a poor biosensor performance. The high- aqueous environment (Fig. 9). Further increase in pH of est DNA biosensor response was obtained when Na ions the DNA hybridisation medium, the arowana DNA bio- were added to the DNA hybridisation phosphate buffer sensor response increased abruptly at pH 7.0, after which because of their small size and strong affinity towards the a sharp decline in DPV current was discernible as the pH DNA phosphodieter bond. environment changed to basic condition due to the The concentration of NaCl and Na-phosphate buffer irreversible denaturation of DNA in the higher pH range (pH 7.0) must also be optimised to provide an optimal [23, 24, 31–33]. Since maximum DPV response was ac- ionic strength for hybridisation buffer. Figure 11b indi- quired at a neutral pH, the next electrochemical evaluation cates that ionic strength of below and above 2 M could of arowana DNA biosensor response was maintained at pH not overcome the high electrostatic repulsion between 7.0 using 0.05 M of Na-phosphate buffer. DNA strands. About 0.05 M of Na-phosphate buffer (Fig. 11a) and 2 M of NaCl were found to provide the optimum Fig. 9 The DPV response of arowana DNA biosensor based on AcMP- 2+ + + 3+ AuNP composite modified carbon SPE between pH 5.5 and pH 8.0. The Fig. 10 The effect of Ca ,Na ,K , and Fe ions in the DNA DPV measurement was conducted in 0.05 M K-phosphate buffer (pH 7.0) hybridisation buffer (0.05 M Na-phosphate buffer at pH 7.0) on the at 25 °C and scan rate of 0.5 V/s versus Ag/AgCl reference electrode DPV response of arowana DNA biosensor i / µA Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 8 of 10 ionic strength for the assay of arowana target DNA with Determination of Arowana Fish Gender with DNA maximum biosensor performance. Optimum hybridisa- Biosensor tion buffer conditions in terms of pH, buffer capacity, and The developed electrochemical DNA biosensor has been ionic strength would allow DNA hybridisation reaction to validated with the standard PCR-based method to deter- occur at the most minimum steric hindrance [30]. mine the gender of Asian arowana fish. With the results The optimised DNA biosensor was then used for the de- tabulated in Table 2, both methods provided the same re- tection of a series of arowana cDNA concentrations be- sult for the gender determination of arowana fish. This in- −12 −2 tween 1.0 × 10 and 1.0 × 10 μM. The DNA biosensor dicates that the proposed DNA biosensor can be used for −18 showed a wide linear response range from 1.0 × 10 to accurate determination of arowana gender in a simple and −8 2 1.0 × 10 M(R = 0.99). The limit of detection (LOD) ob- fast way. −18 tained at 1.0 × 10 M was calculated based on three times the standard deviation of biosensor response at the re- Conclusions sponse curve approximating LOD divided by the linear cali- The electrochemical DNA biosensor developed in this study bration slope. The homogeneous AcMP particles size demonstrated good sensitivity, wide linear response ranges, within micrometre range exhibited a significant influence and low detection limit in the determination of arowana tar- on the DNA biosensor sensitivity and reproducibility get DNA. In addition, the DNA biosensor showed a good (RSD =5.6%).The largebinding surfaceareaof the immo- response towards arowana cDNA, which implies that the bilised NAS-functionalised AcMPs permitted a large num- electrochemical DNA biosensor could be used to success- ber of DNA molecules to bind covalently to the electrode fully detect the arowana DNA segments. The developed surface, thereby increasing the DNA biosensor analytical arowana DNA biosensor can be further redesigned into a performance with respect to dynamic linear range and de- point-of-use device prototype that offers a great potential tection limit of the arowana DNA biosensor (Fig. 12). Fig. 11 The arowana DNA biosensor response trends as the a Na- phosphate buffer concentration and b ionic strength of the hybrid- Fig. 12 The arowana DNA biosensor response curve (a) and linear isation buffer varied from 0.002–0.100 M and calibration range (b) and the DPV voltammogram (c) obtained using −18 −2 1.52–5.50 M, respectively 1.0 × 10 to 1.0 × 10 μM cDNA at pH 7.0 Rahman et al. Nanoscale Research Letters (2017) 12:484 Page 9 of 10 Table 2 A comparison between DNA biosensor and PCR method in the gender identification of arowana fish using fish tissue samples No Sample DNA biosensor method PCR method Current (μA) RSD Baseline ± SD t test Gender 1 227 1.479 ± 0.138 9.360 1.885 ± 0.10 5.042** FF 2 231 2.315 ± 0.149 6.453 1.885 ± 0.10 7.184** MM 3 232 2.627 ± 0.185 7.053 1.885 ± 0.10 7.219** MM 4 233 1.829 ± 0.158 8.643 1.885 ± 0.10 1.117 F F 5 236 2.021 ± 0.169 8.372 1.885 ± 0.10 1.387 F F 6 417 2.947 ± 0.215 7.291 1.956 ± 0.06 10.412** MM 7 437 2.779 ± 0.089 3.217 1.956 ± 0.06 22.126** MM 8 450 1.964 ± 0.122 6.215 1.956 ± 0.06 0.093 F F 9 530 2.500 ± 0.232 9.264 1.956 ± 0.06 4.542** MM 10 531 2.581 ± 0.195 7.556 1.956 ± 0.06 6.544** MM 11 537 2.001 ± 0.189 9.441 1.993 ± 0.12 0.124 F F 12 521 1.672 ± 0.043 2.600 1.993 ± 0.12 6.599** FF 13 524 2.774 ± 0.102 3.678 1.993 ± 0.12 10.775** MM 14 525 1.359 ± 0.075 5.512 1.993 ± 0.12 7.377** FF 15 526 2.953 ± 0.169 5.731 1.993 ± 0.12 10.857** MM M male, F female Y X ** –DPV current significantly higher than the baseline current (obtained from PBS buffer alone) indicates male fish; ** –DPV current significantly lower than the baseline current indicates female fish, critical value t = 2.78 (p = 0.05, 95%) for the application in the fish culture for early identification Received: 4 April 2017 Accepted: 27 July 2017 of arowana gender and colour, which is economically ad- vantageous in fishery and aquaculture sectors. 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Nanoscale Research LettersSpringer Journals

Published: Aug 10, 2017

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