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An electrochemical biosensor for the rapid genetic identification of Musang King durian

More than 200 different cultivars of durian exist worldwide but Durio zibethinus or Musang King (MK) is the most premium and prized durian fruit among the recommended varieties. Early identification of this premium variety is critical to protect from non-authentic MK durian cultivars. However, the MK variety's morphological traits are nearly identical to other varieties. Currently, the identification of durian varieties is mostly performed via evaluation of leaf shape, fruit shape, aroma, taste and seed shape and this requires trained personnel for the morphology observation. To enable the rapid identification of the MK variety, PCR amplification of ten durian varieties using six gene candidates from the chloroplast genome was first performed to obtain DNA probes that were specific to the MK durian variety. PCR amplification of ten durian varieties using primers designed confirmed that the nadhA gene sequence showed an obvious difference in the MK variety from other durian varieties. The unique sequence of MK was used as a DNA probe to develop an electrochemical biosensor for the direct identification of the MK durian variety. The electrochemical biosensor was based on the hybridization response of the immobilized DNA probe with the target DNA from the MK variety and was monitored via differential pulse voltammetry technique. Under optimal conditions, the DNA electrochemical biosensor showed a low detection limit at 10% of MK genomic DNA concentration with a wide linear calibration range of 0.05–1.5 µM (R2 = 0.9891) and RSD value of 3.77% (n = 3). The results of the developed DNA biosensor provide high promise for the development of portable sensors employed in the determination of MK variety in the field.

www.nature.com/scientificreports/ to obtain new cultivars from one breeding generation 7 . To shorten the period of selection and to make it costeffective for durian breeding, it is essential to have a durian varieties identification tool for progeny selection. However, identification of MK variety is challenging due to some varieties showing similar morphological characteristics, which are difficult to distinguish and not applicable to opened durian fruits without shells in the markets. The distinctive odours of different durian cultivars, including MK have also been biochemically studied and characterized as a complex suite of odor-active compounds including sulfur volatiles, esters, alcohols, and acids 8,9 . This, therefore, results in a greater demand for quality planting materials of specified variety. The determination of the durian identity can be ideally conducted by the integration of morphological and molecular characterizations. Currently, the identification of durian varieties is mostly performed via evaluation of leaf shape, fruit shape, aroma, taste and seed shape 7, 10 and could only be by trained personnel. In 2018, a draft whole-genome assembly of the MK cultivar was reported in Singapore, providing a piece of useful information for durian agronomy 11 . The complete reported genome of MK may aid in the identification of cultivar-specific sequences, particularly Single nucleotide polymorphism (SNPs) associated with crucial cultivar-specific features (such as flavor, texture, and odor), and it may enable the molecular barcoding of distinct durian cultivars for rapid quality control and identification.
However, SNP marker validation entails labor-intensive and costly 12 . Furthermore, SNP genotyping analysis of one sample at a time is extremely expensive and time-consuming 13 . Moreover, SNPs are less polymorphic than other molecular markers because of their biallelic nature 13 . The other existing DNA markers technology, such as simple sequence repeats (SSR) [14][15][16] , inter-simple sequence repeat (ISSR) 17,18 , and random amplified polymorphic DNA (RAPD) markers 19,20 have been deployed in the identification of selected durian varieties. Molecular markers are nucleotide sequences that represent variation in nucleotide sequences across individuals of a species and are located at a known position on the chromosome. DNA markers are widely utilized in practical agriculture monitoring, mostly due to their reliability, stability, reproducibility, efficiency, and cost-effectiveness. In 2018, the simple sequence repeats (SSR) marker was employed as an alternative technique to study genetic variation among Durian varieties in Malaysia owing to its co-dominant inheritance, multi-allelic nature, and high reproducibility compared to other markers 13,16 . However, the development process of SSR marker is quite lengthy and expensive, and throughput is low due to automation and output data management limitations 21 .Though RAPD and ISSR markers are a suitable alternative to provide useful information for the identification of plant variety and cultivar, general genetic diversity assessment, population genetic structure and plant protection, however, the dominant character of these markers considers them inappropriate for DNA fingerprinting and genetic variation analysis 22 . Notably, the information derived from dominant genetic markers is less informative than that derived from co-dominant genetic markers, as codominant inheritance of markers can differentiate between homozygous and heterozygous states based on the generated alleles but not dominant inheritance of markers. Furthermore, some dominant genetic markers are known to suffer from poor reproducibility 13,23 , raising concerns about the feasibility and reliability of using dominant genetic markers for downstream applications.
With the advent of DNA biosensor technology, the gap in durian variety identification can be bridged. Therefore, in this work, we attempted to develop an electrochemical DNA biosensor, which can be useful to differentiate and to distinguish the identities among the durian cultivars. This technique is highly sensitive, simple, and low cost for MK durian variety determination with good accuracy. Electrochemical DNA biosensors have been widely adopted in agriculture, particularly in disease diagnosis, due to their potential to give higher sensitivity, faster analysis, portability, and lower cost than traditional technologies 24 . The selection of the right immobilization method and the suitability of matrix type for DNA probe immobilization are imperative owing to producing a high-performance DNA biosensor. The selection of DNA probe is based on DNA barcoding for plants technique, wherein genes encoding a mitochondrial cytochrome oxidase (COI, coxI) subunit were extensively employed 25,26 . The development of the DNA barcoding technique allows for high rapididentification of plant varieties and cultivars efficiently 13 . The chloroplast genome in plants is considered a potential candidate for barcoding genes due to conserved gene order, high copy number and simple amplification by PCR 13,27 .
In this research, the six genes candidate from DNA barcoding for plants were deployed by PCR amplification of ten durian varieties to identify DNA sequences that are unique to the MK variety but not found in other durian varieties and to develop as DNA probes. The DNA probe specific to the MK durian variety was used further to develop an electrochemical biosensor for the direct identification of the MK durian variety. The biosensor was constructed from the carbon-paste screen-printed electrode (SPE), which was modified by depositing with gold nanoparticles (AuNPs) and acrylic microspheres (AcMPs) containing succinimide functional groups as the matrix for DNA probe immobilization. The succinimide functional group of the acrylic microspheres become a linker to immobilize aminated DNA probe via covalent bonds 28,29 . The acrylic microspheres provide the advantages of small size and serve a large surface area to volume ratio for DNA probe immobilization on the surface, thus avoiding any barriers to the diffusion of reactants and products 30 . The overall electrode design provides a wide surface area for chemical reactions and excellent surface immobilization properties, which can significantly increase the effectiveness of the electrochemistry detection assay [31][32][33] . Thus, acrylic microspheres (AcMPs) based DNA biosensors have the potential to serve as a promising platform for the development of rapid, sensitive, specific, and portable diagnostic tools for DNA detection [34][35][36] .

Results and discussion
DNA probe determination of 'Musang King'. The sequencing analysis of ten durian varieties showed that there are some unique regions found in loci nadhA gene sequence and had obvious comparison among ten durian varieties in which D99, D145, MDUR88, D175, MDUR78, D24, D168, D200, and MDUR79 durian varieties sequences containing sequence lesion of 17 bases but not in MK DNA sequence (Fig. 1). This comparison implies that the unique sequence in the MK DNA sequence can be used as a DNA probe to differentiate between www.nature.com/scientificreports/ MK with the other durian varieties via electrochemical biosensor. Whereas the other five gene candidates did not show any significant differences among the ten durian varieties sequence (Fig. S1) DNA barcode candidates are based on chloroplast genome that provides some advantages such as simple and stable genetic structure, it is haploid, undergoes no (or very rare) recombination, it is generally uniparentally transmitted, and universal primers that are suitable to use for amplifying target sequences 37 . Additionally, chloroplast genomes are high conservation over nuclear and mitochondrial genomes. Therefore, partial chloroplast genome sequences are preferred and suitable to use for phylogenetic studies and species/varieties identification and discrimination with similar morphology characteristics [38][39][40][41][42][43] . DNA barcoding has been deployed in prior studies owing to its effectiveness in plant variety and cultivar identification. The chloroplast genome is considered a promising candidate for barcoding genes in various land plants due to its advantages, such as conserved gene order, high copy number, and ease of amplification by PCR 13 . The vast of studies on plant barcoding employs one or a few plastid areas, including the protein-coding rbcL and matK regions, the noncoding spacer trnH-psbA, and ITS regions. For instance, the three chloroplast genomes (ITS2 and cp gene matK and rpl32-trnL (UAG)) were successfully identified and discriminated 12 commercial varieties of kiwi fruit 44 . Likewise, Emirati date palm cultivars were able to discriminate using the chloroplast intergenic spacer psbK-psbI 45 . In Xinjiang, China, Artemisia L. varieties were identified using the chloroplast genome of ITS region which revealed the highest identification efficacy 46 .
The response of the biosensor towards target DNA concentrations. Under the optimal conditions, the analytical performance of the DNA biosensor was examined using the immobilized DNA probe by the response of the AcMP-AuNP-modified carbon SPEs electrode towards an increasing concentration of synthetic target DNA (1 µM, 1.5 µM, 0.1 µM, 0.05 µM, and 0.01 µM), non-complementary (NC) DNA and no target (NT) at a scan rate of 0.04 V s −1 , the results were illustrated in Fig. 2. The current response was increased steadily with increasing target DNA concentration at the electrode in experiment (a). The highest peak was 1.5 µM followed by 1.0 µM, 0.1 µM, 0.05 µM, 0.01 µM, and NC. The DPV peak reading of all concentrations was in the range of − 0.55 V to − 0.45 V. This indicates more DNA duplex was formed on the electrode through DNA hybridization reactions followed by anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) intercalation. Additionally, significant current differences were observed in the DPV peak from all concentrations, implying that the DNA probes were successfully immobilized onto the AcMP via covalent bonds between succinimide group of AcMP and amine functional group of the DNA probe 28,29 . The concentration of 1.5 µM was the highest peak, indicated hybridization where intercalation of AQMS had occurred in double-stranded DNA (dsDNA) formed in the microsphere surface with the current peak value of 3.34 µA. However, the DPV peak of NT was the lowest of other concentrations due to no DNA hybridization reaction occurring, indicating there was no specific adsorption of AQMS redox indicator onto AcMP-AuNP-based DNA modified carbon SPEs electrode 47 .
The DNA biosensor responses with five different concentrations of synthetic target DNA and no target that has been hybridized on DNA probe immobilized on AcMPs are shown in Fig. 2b. DNA biosensor response increased proportionally with increasing concentrations of synthetic DNA target immobilised. This suggests that the capacity of immobilized DNA probe to hybridize with complementary of five different synthetic target DNA concentrations has increased with an increase in DNA target attached to the AcMP-AuNP. The linear response range of the DNA biosensor from different concentrations of synthetic target DNA demonstrates that the oxidation peak current of DNA increased linearly with increasing synthetic target DNA concentration and revealed a satisfactory correlation coefficient value of R 2 = 0.9891, and the linear equation was expressed y = 2.1006x − 0.193 with a low detection limit of 0.01 µM (Fig. 2c). The large binding surface area of the AcMPs enabled a large number of DNA molecules to bind covalently to the AcMPs via succinimide functional groups, ultimately enhance the analytical performance of the DNA biosensor in terms of dynamic linear range and detection limit 30 .
The specificity of the biosensor response. Figure 3a shows the DPV response of DNA biosensors with different Durian varieties (MK, D24, MDUR88, MDUR78, MDUR79, D168, D200, D145, D99, D175) and blank (without DNA) under optimum conditions. The DPV peak near − 0.55 V indicates the oxidation of AQMS that has been intercalated into the dsDNA formed from MK DNA probe hybridized with MK complementary DNA. This result revealed that the designed DNA probe from MK is highly specific to MK. MK variety yielded the highest current response as expected and demonstrated that the immobilized MK DNA probe was selective  The sensitivity of the biosensor. To investigate the sensitivity of the biosensor, increasing amounts of MK genomic DNA were added to triplicate reactions containing a decreasing amount of MDUR88 genomic DNA. The genomic DNA mixture containing 0%, 10%, 20%, %, 40%, 60%, 80%, and 100% of MK genomic DNA. This is for the purpose of determining the biosensor response towards MK genomic DNA purity as to whether it is derived solely from clone or hybrid. Figure 4a represents the DPV of different genomic DNA concentrations of MK (0%, 10%, 20%, 40%, 60%, 80%, and 100%) in the present of MDUR88 genomic DNA. The current response of this proposed biosensor clearly increased with the increase of the target concentrations from 0 to 100%, followed by intercalation of AQMS in between the dsDNA ( Fig. 4a and b). The DNA biosensor showed a good linear relationship with the percentage of MK gDNA (Fig. 4c), and the linear equation was expressed y = 3x − 0.1 (R 2 = 0.9712) with a detection limit down to 10% of MK gDNA for sensitivity analysis. The reproducibility of the DNA biosensor was performed with 100% of MK gDNA. DNA biosensors gave satisfactory reproducibility results with 3.77%. The DPV peak reading of all concentrations was at − 0.55 V. This suggests an increase in hybridization between the immobilized DNA probes with the target DNA when the DNA concentrations increased.
Biosensor response validation with real durian DNA samples. In order to prove the effectiveness of the biosensor in determining and distinguishing the Durian MK variety from other Durian varieties, a total of 27 different durian field samples were analysed with the electrochemical DNA biosensor. Figure 5 illustrates the effect of different durian DNA samples on the biosensor response. Based on the DPV diagram in Fig. 5a-d, all field samples were identified as MK variety except for DS2 and DS18 from Sintok, Kedah, which yielded current as low as the non-MK control sample MDUR88. As shown in Fig. 5e, the mean of 24 MK samples is 0.93, and the electrochemical response of the biosensor for all samples was comparable with the MK standard, which revealed the current of 0.93 ± 0.25 µA except for MKS2 and MKS18. These results from the electrochemical DNA biosensor have been validated with the standard PCR-based method and followed by sequencing to determine the 27 durian field samples. PCR amplification of 27 durian samples produced 300 bp amplicon except for DS2 and DS18 samples (Fig. S2). With the results tabulated in Table 1, both methods provided the same result for

Conclusion
In this study, an electrochemical DNA biosensor was successfully developed with high specificity, good sensitivity, wide linear response ranges, and low detection limit in the determination of MK durian. Furthermore, the electrochemical DNA biosensor showed a good response to the MK DNA target, which implies that the biosensor is sensitive and has high selectivity to determine MK variety. Moreover, the developed MK DNA biosensor can assist farmers for early identification of MK durian variety in order to avoid confusion in the propagation system, which is economically advantageous in agriculture sectors. Furthermore, farmers can benefit from the use of biosensors for the identification of MK durian variety since it can help them to ensure the authenticity of MK, which is crucial for export activities to foreign nations. Early determination and confirmation of MK's authenticity at the seedling stage is also critical to ensuring that farmers do not suffer significant losses due to their large investment in Mk durian trees. Employing DNA biosensors to identify MK durian variety also has the advantage of allowing farmers or dealers to obtain proof of certification for export permission to an international market. The identification of MK is also not only beneficial to farmers, but it can also protect the rights of consumers in meeting the high demand for trees or fruit as MK is a durian variety that has a high global market.   ATT GAC GGC TCA AGNACA ACC GTA TAG AGT TTT GGC TAT CCT TTG TAT AGA TTCC CAT ACA TGA  TAG ATT CCC ATA CAT GTA TTG CCA AAC CAA ACG GGG GAT TGA ACA AAA AAA TGA GTG GAT GGT  TAG GAA CAC CAA AAA   Yes  Yes   2  DZ4   TCA TTT TGA CGG GCT CAA GAT CAA CCG TAT AGA GTT TTG GCT ATC CTT TGT ATA GAT TCC CAT  ACA TGA TAG ATT CCC ATA CAT GTA TTG CCA AAC CAA ACG GGG GAT TGA ACA AAA AAA TGA GTG  GAT GGT TAG GAA CAC  The DNA sequences of PCR products from ten durian varieties were aligned using Clustal Omega (https:// www. ebi. ac. uk/ Tools/ msa/ clust alo/) and unique DNA sequences that can differentiate MK from the other nine varieties were identified and used as a DNA probe in DNA biosensor development for MK determination.
Design of DNA probe for biosensor. DNA probe, target DNA and non-complementary DNA (Table 3) were designed based on the bioinformatics study of nadhA gene (based on unique DNA sequence revealed from sequence alignment of 10 durian varieties).  NTGA CGG CTC AAG ATC AAC CGT ATA GAG TTT TGG CTA TCC TTT GTA TAG ATTCC CAT ACA TGA  TAG ATT CCC ATA CAT GTA TTG CCA AAC CAA ACG GGG GAT TGA ACA AAA AAA TGA GTG GAT GGT  TAG GAA CAC CAA AA   Yes  Yes   26  DS39   TTG ACG GCT CAA ANACA ACC GTA TAG AGT TTT GGC TAT CCT TTG TAT AGA TTCC CAT ACA TGA  TAG ATT CCC ATA CAT GTA TTG CCA AAC CAA ACG GGG GAT TGA ACA AAA AAA TGA GTG GAT GGT  TAG GAA CAC     Fabrication of DNA biosensor. Two mg of acrylic microspheres were weighed in a microcentrifuge tube and 300 µL of 1 µM animated DNA probes was added to immobilize the DNA onto the microspheres for 24 h at 4 °C. The DNA /AcMPs were collected after 24 h by centrifuging at 1000 rpm for 8 min. The supernatant (which contains DNA probes) was kept for future use in DNA immobilization. The microspheres with K-phosphate were washed several times, and the wash solution was discarded. Fresh 0.05 M K-phosphate (pH 7.0) buffer was added to re-suspense the DNA microspheres. One mg of gold nanoparticles (AuNPs) was suspended in 300 µL ethanol and 10 µL of AuNPs suspension were drop-coated onto C-SPE. The electrode AuNPs/SPE is then left to dry at room temperature for 1-2 h. AcMPs immobilized with DNA probes were pipetted (8 µL) and dropped it onto AuNPs/CSPE and stored at 4 °C until dry for 24 h. 10 µL of solution containing DNA target, 0.05 M of Na-phosphate buffer and 1.0 mM AQMS was dropped onto the electrode surface and incubated for 40 min for hybridization of the probe with the target. The electrode was then rinsed several times with 0.05 M K-phosphate buffer. An amount of 100 µL fresh 0.05 M K-phosphate was dropped onto the electrode surface, and the DPV was scanned at the potential range of (− 0.8 V to − 0.2 V) of the electrode using CSPE as a working electrode, platinum as the counter electrode and Ag as a reference electrode. The AQMS peak should appear at approximately − 0.55 V. All the experiments were performed in triplicate. The SPE construction and design mechanism of the electrochemical DNA biosensor is illustrated in Fig. 6. Sensitivity of biosensor system. The biosensor experiment was performed on different concentration of MK percentage (100%, 80%, 60%, 40%, 20%, 10%, and 0%), where genomic DNA of MK variety was mixed with genomic DNA from MDUR88 variety. In 1 µL of DNA sample was containing total of 10 ng MK and MDUR88. The combination of MK and MDUR88 gDNA used in this study were shown in the Table 4.

Field samples test. A total of 27 durian samples were collected from five different regions in Peninsular
Malaysia as shown in Table 5. The optimised DNA biosensor was then used for the determination MK gDNA via DPV method.
Validation. The biosensor performance of 27 MK samples was validated via PCR based-method using designed primers from nadhA gene sequence as shown in Table 6. The reaction mixture and PCR program used in validation were similar as used in the DNA probe selection for MK biosensor development. Amplicons were confirmed using gel electrophoresis. The PCR products were then purified and outsourced for DNA sequencing at 1st BASE, Malaysia. From the DNA sequence of PCR product, unique sequence of MK was identified, thus validate the biosensor performance in identifying MK field samples.

Data availability
The datasets generated and analysed during the current study are available in the European Variation Archive (EVA) at EMBL-EBI under accession number PRJEB55857 (https:// www. ebi. ac. uk/ eva/? eva-study= PRJEB 55857).
Additional raw data will be available upon request.