- Nano Express
- Open Access
A Highly Sensitive Electrochemical DNA Biosensor from Acrylic-Gold Nano-composite for the Determination of Arowana Fish Gender
© The Author(s). 2017
- Received: 4 April 2017
- Accepted: 27 July 2017
- Published: 10 August 2017
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 acid redox probe as an electroactive oligonucleotide label (Table 1). A low detection limit at 1.0 × 10−18 M with a wide linear calibration range of 1.0 × 10−18 to 1.0 × 10−8 M (R 2 = 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.
- DNA biosensor
- Electrochemical biosensor
- Acrylic microspheres
- DNA hybridization
- Arowana DNA
Asiatic arowana (Scleropages formoss), a freshwater fish,  is widely distributed over the countryside of Southeast Asia region such as Malaysia, Singapore, Thailand, Indonesia, Cambodia, Vietnam, Laos, Myanmar and the Philippines. In addition, the arowana fish is also found in Australia and New Guinea [1–4]. It is popularly known as dragonfish, Asia bonytongue, kelisa, or baju-rantai [5, 6]. It is still surviving as a primitive fish species from the Jurassic era [7, 8]. The Chinese and Asian people considered it as a symbol of good luck and happiness, along with many other cultures . Generally, the arowana is around 7 kg weight and 1 m long in their mature age . This ornamental fish possesses attractive colours and morphology and can be identified by its distinctive physical features, such as comparatively long in body size, a large pectoral fin, and the dorsal and anal fins are positioned far back on the body. There are three main colour varieties, i.e. golden, red, and green of closely related freshwater fish within the Asian arowana species. There are also several other distinct species derived from different parts of the Southeast Asia and are regional to many river systems .
Due to its high popularity and great demand in ornamental purposes, Asian arowana has been fiercely hunted for profits , and results in a rapid decline of its population. Considering its high demand in ornamental industry, the over-exploitation of natural populations, and the rarity of natural habitats due to changes in the living environment, Asian arowana has been classified as an endangered species threatened with extinction since 1980 by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and has recently listed as endangered by the 2006 IUCN Red List [1, 3, 8, 10, 11]. However, the commercial trading of this endangered species is prohibited under CITES except in certain countries, e.g., Indonesia, Singapore, and Malaysia. [2, 3, 12]. There are a number of CITES registered cultivators in Asia actively carrying out farming and trading of arowana fish [2, 12]. This Asian freshwater fish consists of geographically isolated strains, and it is the only member of the species with different colour varieties that is based on different geographic distributions throughout the rivers of Southeast Asia. The species distribution is now far more widespread, which extends to the Nile River of Africa, the Amazon River of South America, Australia, and New Guinea [1, 4, 8].
Among the different colours of Asiatic arowana, red and golden arowana fishes are the most expensive and popular ornamental pets in the hatchery industry compared to black, green, silver, and others colour varieties [1, 5, 10, 13]. The egg thievery phenomenon of Asiatic arowana is atypical compared to other fish species. In general, arowana fishes get mature at the age of 3–4 years, and they lay only a few eggs (30–100) [14, 15] of extra-large size (around 1 cm in diameter) . Interestingly, the fertilised eggs and larvae are then protected and grown up in the mouth of male arowana fishes, and they show high parental care. To identify the gender based on visual observation of the baby arowana is difficult because there is no distinctive phenotypic organ of sexual dimorphism [14, 15]. Only one of the parents (presume to be the male) can be identified as the offspring are harvested from his mouth. The other parent cannot be identified from among a number of potential parents .
Usually, the hobbyists keep the baby arowana fish for their ornamental purposes in the aquarium as well as for cultivation in the fish farm. However, all types of juvenile arowana fishes are sold at the same price, because of the lack of assistive technology for gender and colour variety differentiation. Until the present time, there is no established method published to identify the gender and colour of arowana fishes at their juvenile stage. Instead, hundreds of studies have been carried out using DNA analysis based on genetic structure and biography of arowana fishes in the attempt to identify the gender and colour at their early age. Traditional method based on body size and mouth cavity estimations can only be made at around 3 months of age of baby arowana for gender and colour identifications . However, this conventional visual examination method is time-consuming and often provides inaccurate result. On the other hand, the widely used standard methods based on DNA sequencing, i.e., polymerase chain reaction (PCR) and gel electrophoresis are labour-, time-, and resource-demanding. An alternative algorithm of inventive problem solving (ARIZ) method was previously employed for the detection of arowana gender detection . ARIZ is an alternative tool for gender detection, containing nine different parts and a total of 40 complex steps. It requires a very long time to learn and practice and demands highly experienced personnel to operate. For example, the application of ARIZ in various engineering systems has been employed, but most of the cases did not cover all the requirements and processes of ARIZ.
In this research, acrylic polymer microspheres modified with succinimide functional groups via N-acryloxysuccinimide (NAS) moieties was used as the matrix for DNA probe immobilisation. As previously reported by Chen and Chiu 2000 and Chaix et al. 2003 [19, 20], the succinimide functional group can react with amine functional groups to form a covalent bond. The incorporation of NAS functionality into acrylic microspheres for DNA microbiosensor application provides advantages of a simple preparation method where the spheres can be synthesised and functionalised via a one-step procedure using photopolymerisation in a short duration (several minutes). In addition, the microspheres have the advantage of small size and provide a large surface area for DNA probe immobilisation, thus reducing the barrier to diffusion for reactants and products. This enables the improvement in the biosensor performance in terms of shorter response times and wider linear response range, which will be demonstrated in the work reported here.
In this study, an electrochemical DNA biosensor method, which is highly sensitive, simple, easy-to-fabricate, and low cost, is proposed for juvenile arowana fish gender determination with high accuracy. The DNA biosensor was built from a carbon screen printed electrode (SPE) modified with colloidal gold nanoparticles (AuNPs) and polyacrylate microspheres functionalised with NAS functional group. The AuNPs were immobilised onto the carbon SPE surface via electrostatistic interaction and played an important role in enhancing the electrode conductivity and facilitating the electron transfer, while the acrylic microspheres (AcMPs) were directly deposited onto the AuNP-modified SPE via physical adsorption. Aminated DNA probe of arowana was then covalently attached to the immobilised AcMP-AuNP composite at the exposed succinimide group of AcMPs. Probe-target hybridisation was detected with anthraquinone redox label via differential pulse voltammetry (DPV). The incorporation of small and uniform size of AcMPs was able to hold a large DNA-loading capacity and enhancing the sensitivity and detection limit of the electrochemical arowana DNA biosensor.
Apparatus and Electrodes
All the electrochemical measurements were performed with DPV using Autolab PGSTAT 12 potentiostat/galvanostat (Metrohm) at 0.02 V step potential within the potential window of −1.0 V to −0.1 V. SPE from Scrint Technology Co Malaysia modified with AcMPs and AuNPs was used as the working electrode. A rod-shaped platinum (Pt) electrode and an Ag/AgCl electrode filled with 3.0 M of KCl internal solution were used as auxiliary and reference electrodes, respectively. Elma S30H sonicator bath was used to prepare homogeneous solutions.
2–2-Dimethoxy-2-phenylacetophenone (DMPP) was purchased from Fluka. 1,6-Hexanediol diacrylate (HDDA), n-butyl acrylate (nBA), and Au (III) chloride trihydrate were supplied by Sigma-Aldrich. The colloidal AuNPs was synthesised according to the method reported by Grabar et al. (1995). Sodium dodecyl sulphate (SDS) and NaCl were obtained from Systerm. NAS and anthraquinone-2-sulfonic acid monohydrate sodium salt (AQMS) were procured from Acros. Milli-Q water (18 mΩ) was used to prepare all the chemical and biological solutions. Stock solution of DNA probe was diluted with 0.05 M of K-phosphate buffer (pH 7.0) while complementary DNA (cDNA) and non-complementary (ncDNA) solutions were prepared with 0.05 M of Na-phosphate buffer at pH 7.0 containing 1.0 mM of AQMS. The K-phosphate buffer facilitates 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 . Briefly, a mixture of 450 μL of HDDA, 0.01 g of SDS, 0.1 g of DMPP, 7 mL of 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 photocured with UV light for 600 s under a continuous flow of N2 gas. The resulting poly(nBA-NAS) microspheres were then collected by centrifugation at 4000 rpm for 30 min followed by washing in K-phosphate buffer (0.05 M, pH 7.0) for three times and left to dry at ambient temperature.
Fabrication of DNA Biosensor Using Acrylic Microspheres
Optimization of Electrochemical Arowana DNA Biosensor
The DNA electrodes modified with the respective AcMP, AuNP, and AcMP-AuNP composite were used in the cDNA (5 μM) and ncDNA (5 μM) testing with DPV electroanalytical method in the presence of 1 mM of AQMS and 2 M of NaCl at the scan rate of 0.5 V/s versus Ag/AgCl reference electrode. DNA probe immobilisation duration was determined by separately soaking nine units of AcMP-AuNP-modified SPEs in 300 μL of 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 buffer 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 μL of 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 response at 4, 25, 40, and 50 °C for an experimental period of 5–90 min in the measuring buffer using DPV technique. 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 positively charged ions (i.e. Ca2+, Na+, K+, and Fe3+ ions) on the electrochemical arowana DNA biosensor response was carried out by adding CaCl2, NaCl, KCl, and FeCl3 into 0.05 M of Na-phosphate buffer (pH 7.0) prior to DNA hybridisation reaction and DPV measurement. Ionic strength of the hybridisation buffer was optimised by varying the Na-phosphate buffer and NaCl concentrations from 0.002–0.1000 M to 1.52–5.50 M, respectively. The linear calibration curve of the arowana DNA biosensor was then established through quantitative measurement of a series of cDNA concentrations from 1.0 × 10−18 to 2.0 × 10−2 μM via DPV method. All the experiments were performed in triplicate.
DNA Extraction and Arowana DNA Analysis
A total of 15 arowana fish tissue samples were kindly provided 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 ambient conditions before kept in the freezer at −20 °C. Arowana DNA from each tissue sample (35–40 mg each) was then separately extracted using QIAquick PCR Purification kit (Manchester, UK) according to the manufacturer’s protocol and stored at −20 °C when not in use. PCR amplification 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 arowana DNA extracts were also analysed by the electrochemical DNA biosensor to determine the gender. The DPV responses obtained were compared with the baseline current obtained without the presence of arowana DNA. A t test was applied to determine significant difference between the DNA biosensor response and baseline current at 4 degrees of freedom and 95% confidence level. The DNA biosensor response obtained at significantly higher than the baseline current indicated a male arowana fish was detected and vice versa.
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 demonstrated larger peak potential separations of more than 0.059 V (Fig 4). This implies that the electron transfer process at the electrode surface is slow [22, 25, 26], probably due to the resistance created by the presence of AcMP material covering the electrode surface.
Determination of Arowana Fish Gender with DNA Biosensor
Sequences of oligonucleotides utilised in the present investigation
5'-AAT TCA AGG GAA CTG ATG ACT CTA (AmC7)
5'-TAG AGT CAT CAG TTC CCT TGA ATT
5'-CGA GCG ACG TGA GCT TAG CTG CGC
A comparison between DNA biosensor and PCR method in the gender identification of arowana fish using fish tissue samples
DNA biosensor method
Baseline ± SD
1.479 ± 0.138
1.885 ± 0.10
2.315 ± 0.149
1.885 ± 0.10
2.627 ± 0.185
1.885 ± 0.10
1.829 ± 0.158
1.885 ± 0.10
2.021 ± 0.169
1.885 ± 0.10
2.947 ± 0.215
1.956 ± 0.06
2.779 ± 0.089
1.956 ± 0.06
1.964 ± 0.122
1.956 ± 0.06
2.500 ± 0.232
1.956 ± 0.06
2.581 ± 0.195
1.956 ± 0.06
2.001 ± 0.189
1.993 ± 0.12
1.672 ± 0.043
1.993 ± 0.12
2.774 ± 0.102
1.993 ± 0.12
1.359 ± 0.075
1.993 ± 0.12
2.953 ± 0.169
1.993 ± 0.12
The electrochemical DNA biosensor developed in this study demonstrated good sensitivity, wide linear response ranges, and low detection limit in the determination of arowana target DNA. In addition, the DNA biosensor showed a good response towards arowana cDNA, which implies that the electrochemical DNA biosensor could be used to successfully detect the arowana DNA segments. The developed arowana DNA biosensor can be further redesigned into a point-of-use device prototype that offers a great potential for the application in the fish culture for early identification of arowana gender and colour, which is economically advantageous in fishery and aquaculture sectors.
This work was supported by funding (XX-2014-005) from Fishery Research Institute (FRI) Glami Lemi, Department of Fisheries, Malaysia and partial funding from Universiti Kebangsaan Malaysia via grants DPP-2016-064. Md Mahbubur Rahman would like to acknowledge a studentship (Skim Zamalah Universiti Penyelidikan) awarded by Universiti Kebangsaan Malaysia.
MR carried out the experiments and drafted the manuscript. LYK and TLL supervised the overall study and polished the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis 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.
- Mohd-Shamsudin MI, Fard MZ, Mather PB, Suleiman Z, Hassan R, Othman RY, Bhassu S (2011) Molecular characterization of relatedness among colour variants of Asian Arowana (Scleropages formosus). Gene 490(1):47–53View ArticleGoogle Scholar
- Kottelat M, Whitten T (1996) Freshwater biodiversity in Asia: with special reference to fish, vol. 343. World Bank PublicationsGoogle Scholar
- Natalia Y, Hashim R, Ali A, Chong A (2004) Characterization of digestive enzymes in a carnivorous ornamental fish, the Asian bony tongue Scleropages formosus (Osteoglossidae). Aquaculture 233(1):305–320View ArticleGoogle Scholar
- Yue GH, Li Y, Lim LC, Orban L (2004) Monitoring the genetic diversity of three Asian arowana (Scleropages formosus) captive stocks using AFLP and microsatellites. Aquaculture 237(1):89–102View ArticleGoogle Scholar
- Tang P, Sivananthan J, Pillay S, Muniandy S (2004) Genetic structure and biogeography of Asian arowana (Scleropages formosus) determined by microsatellite and mitochondrial DNA analysis. Asian Fisheries Science 17(1/2):81–92Google Scholar
- Hu Y, Mu X, Wang X, Liu C, Wang P, Luo J (2009) Preliminary study on mitochondrial DNA cytochrome b sequences and genetic relationship of three Asian arowana Scleropages formosus. International Journal of Biology 1(2):28View ArticleGoogle Scholar
- Bonde N (1979) Palaeoenvironment in the “North Sea” as indicated by the fish bearing Mo− Clay deposit (Paleocene/Eocene), Denmark. Mededelingen van de Werkgroep voor Tertiaire en Kwartaire Geologie 16(1):3–16Google Scholar
- Mu XD, Song HM, Wang XJ, Yang YX, Luo D, Gu DE, Luo JR, Hu YC (2012) Genetic variability of the Asian arowana, Scleropages formosus, based on mitochondrial DNA genes. Biochem Syst Ecol 44:141–148View ArticleGoogle Scholar
- Alfred E (1964) The fresh-water food fishes of Malaya. I. Scleropages formosus (Müller and Schlegel). Fed Mus J 9:80–83Google Scholar
- Yue GH, Liew WC, Orban L (2006) The complete mitochondrial genome of a basal teleost, the Asian arowana (Scleropages formosus, Osteoglossidae). BMC Genomics 7(1):242View ArticleGoogle Scholar
- Greenwood PH, Rosen DE, Weitzman SH, Myers GS (1966) Phyletic studies of teleostean fishes, with a provisional classification of living forms. Bulletin of the AMNH 131(4)Google Scholar
- Fernando A, Lim L, Jeyaseelan K, Teng S, Liang M, Yeo C (1997) DNA fingerprinting: application to conservation of the CITES-listed dragon fish, Scleropages formosus (Osteoglossidae). Aquar Sci Conserv 1(2):91–104View ArticleGoogle Scholar
- Yue G, Ong D, Wong C, Lim L, Orban L (2003) A strain-specific and a sex-associated STS marker for Asian arowana (Scleropages formosus, Osteoglossidae). Aquac Res 34(11):951–957View ArticleGoogle Scholar
- Dawes JA, Lim LC, Cheong L (1999) The dragon fish. Kingdom BooksGoogle Scholar
- Scott D, Fuller J (1976) The reproductive biology of Scleropages formosus (Müller & Schlegel) (Osteoglossomorpha, Osteoglossidae) in Malaya, and the morphology of its pituitary gland. J Fish Biol 8(1):45–53View ArticleGoogle Scholar
- Chang AKW, Liew WC, Orban L (2007) The reproduction of Asian arowana: analysis by polymorphic DNA markers. Aquaculture 272(1):S249Google Scholar
- Suleiman MZ (2003) Breeding technique of Malaysian golden arowana, Scleropages formosus in concrete tanks. Aquaculture Asia 8(3):5–6Google Scholar
- Benjaboonyazit T (2014) Systematic approach to arowana gender identification problem using algorithm of inventive problem solving (ARIZ). Engineering Journal 18(2):13–28View ArticleGoogle Scholar
- Chen J-P, Chiu S-H (2000) A poly (N-isopropylacrylamide-co-N-acryloxysuccinimide-co-2-hydroxyethyl methacrylate) composite hydrogel membrane for urease immobilization to enhance urea hydrolysis rate by temperature swing☆. Enzym Microb Technol 26(5):359–367View ArticleGoogle Scholar
- Chaix C, Pacard E, Elaissari A, Hilaire JF, Pichot C (2003) Surface functionalization of oil-in-water nanoemulsion with a reactive copolymer: colloidal characterization and peptide immobilization. Colloids Surf B: Biointerfaces 29(1):39–52View ArticleGoogle Scholar
- Ulianas A, Heng LY, Ahmad M, Lau H-Y, Ishak Z, Ling TL (2014) A regenerable screen-printed DNA biosensor based on acrylic microsphere–gold nanoparticle composite for genetically modified soybean determination. Sensors Actuators B Chem 190:694–701View ArticleGoogle Scholar
- Ulianas A, Heng LY, Hanifah SA, Ling TL (2012) An electrochemical DNA microbiosensor based on succinimide-modified acrylic microspheres. Sensors 12(5):5445–5460View ArticleGoogle Scholar
- Mohiuddin M, Arbain D, Shafiqul Islam A, Rahman M, Ahmad M, Ahmad M (2015) Electrochemical measurement of antidiabetic potential of medicinal plants using screen-printed carbon nanotubes electrode. Curr Nanosci 11(2):229–238View ArticleGoogle Scholar
- Mohiuddin M, Arbain D, Islam AS, Rahman M, Ahmad M, Ahmad M (2015) Electrochemical measurement of the antidiabetic potential of medicinal plants using multi-walled carbon nanotubes paste electrode. Russ J Electrochem 51(4):368–375View ArticleGoogle Scholar
- Lu T-L, Tsai Y-C (2011) Sensitive electrochemical determination of acetaminophen in pharmaceutical formulations at multiwalled carbon nanotube-alumina-coated silica nanocomposite modified electrode. Sensors Actuators B Chem 153(2):439–444View ArticleGoogle Scholar
- Kalimuthu P, John SA (2010) Simultaneous determination of ascorbic acid, dopamine, uric acid and xanthine using a nanostructured polymer film modified electrode. Talanta 80(5):1686–1691View ArticleGoogle Scholar
- Batra B, Lata S, Sharma M, Pundir C (2013) An acrylamide biosensor based on immobilization of hemoglobin onto multiwalled carbon nanotube/copper nanoparticles/polyaniline hybrid film. Anal Biochem 433(2):210–217View ArticleGoogle Scholar
- Wong EL, Erohkin P, Gooding JJ (2004) A comparison of cationic and anionic intercalators for the electrochemical transduction of DNA hybridization via long range electron transfer. Electrochem Commun 6(7):648–654View ArticleGoogle Scholar
- Zhang W, Yang T, Li X, Wang D, Jiao K (2009) Conductive architecture of Fe 2 O 3 microspheres/self-doped polyaniline nanofibers on carbon ionic liquid electrode for impedance sensing of DNA hybridization. Biosens Bioelectron 25(2):428–434View ArticleGoogle Scholar
- Metzenberg S (2007) Working with DNA: the basics. Taylor & Francis Group, FlorenceGoogle Scholar
- Hames BD, Higgins SJ (1985) Nucleic acid hybridisation: a practical approachGoogle Scholar
- Feng K-J, Yang Y-H, Wang Z-J, Jiang J-H, Shen G-L, Yu R-Q (2006) A nano-porous CeO 2/Chitosan composite film as the immobilization matrix for colorectal cancer DNA sequence-selective electrochemical biosensor. Talanta 70(3):561–565View ArticleGoogle Scholar
- Pan J (2007) Voltammetric detection of DNA hybridization using a non-competitive enzyme linked assay. Biochem Eng J 35(2):183–190View ArticleGoogle Scholar
- Zhu N, Cai H, He P, Fang Y (2003) Tris (2, 2′-bipyridyl) cobalt (III)-doped silica nanoparticle DNA probe for the electrochemical detection of DNA hybridization. Anal Chim Acta 481(2):181–189View ArticleGoogle Scholar