快速检测光学八植物油薄膜生物传感器芯片

Rapid detection of eight vegetable oils on optical thin-film biosensor chipsSulan Bai a ,*,1,Shucheng Li a ,1,Tao Yao a ,Yong Hu a ,Fang Bao a ,Jie Zhang b ,Yaochuan Zhang b ,Shengwei Zhu c ,Yikun He a ,*aKey Laboratory of Genetics and Biotechnology,College of Life Sciences,Capital Normal University,Beijing 100048,PR China bBeijing Vocational College of Agriculture,Beijing 102442,PR China cKey Laboratory of Photosynthesis and Environmental Molecular Biology,The Institute of Botany,Chinese Academy of Sciences,Beijing 100093,PR Chinaa r t i c l e i n f oArticle history:Received 21December 2010Received in revised form 10March 2011Accepted 12March 2011Keywords:AssayVegetative oilsThin-film biosensor chipsa b s t r a c tThe development of new and increasingly sophisticated techniques for the authentication of food products continues to be a challenge in public food safety.The authenticity of vegetable oils has become very important as the surge of counterfeit oil into the market increases and consumer con fidence decreases.The two methods currently used to authenticate vegetable oils are analysis of species-speci fic chemical composition by techniques such as chromatography and mass spectrometry,and DNA-based methods such as microarray or qPCR detection.In this study,we have developed a thin-film biosensor chip-based analytical technique for food authentication.Aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface.Biotinylated PCR amplicons were then hybridized with the probes.After wash and brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate,biotinylated PCR chain perfectly matched with the probes can be visualized as a color change on the chip surface (gold to blue/purple).This method could detect trace amount of species-speci fic DNA from food products and has been demonstrated to be effective with eight different vegetative oils.Our results indicated that this assay is rapid,highly sensitive and speci fic,and can potentially be used as a high throughput detection method.Ó2011Elsevier Ltd.All rights reserved.1.IntroductionThere is a wide variety of vegetable oils available to consumers and their prices mostly depend on the oil ’s quality and purity.Unfortunately,some counterfeit vegetable oils adulterated by illegal operators are often being sold for pro fiteering.Species adulteration of vegetable oil will lead to economic losses and loss of consumer con fidence,and may even negatively impact legitimate operators.Therefore,a reliable method of species authentication for vegetable oils is desirable.Currently there are two major methods for vegetable oil species authentication.The first is the conventional method of chemical constituent analysis to identify the speci fic vegetable oil based on species-speci fic chemical composition.Analytical techniques such as proton transfer reaction mass spectrometry (PTR-MS),nuclear magnetic resonance spectroscopy (NMR),high performance liquid chromatography (HPLC;Luykx &van Ruth,2008),and gas chro-matography (GC;Dourtoglou et al.,2003)are commonly used toanalyze olive oil.However,the chemical composition of oil may differ among different seasons and growing areas.Moreover,it was found that no single known parameter could detect the presence of other oils in olive oil,if their concentration is lower than or equal to 5%(Christopoulou,Lazaraki,Komaitis,&Kaselimis,2004).Conse-quently,chemical analyses may not be suf ficient for oil authenti-cation (Giménez,Pistón,Martín,&Atienza,2010).The second method is based on DNA genetic identi fication and involves the use of polymerase chain reaction (PCR)and molecular markers.PCR technology has been proven to be an invaluable method for species identi fication via ampli fication of species-speci fic genes,which can be employed to identify species composition in food (Rodríguez et al.,2003).Real-time PCR is frequently used for quantitative and qualitative analysis of species-speci fic genes,such as the detection of genetically modi fied soybean DNA in re fined vegetable oils (Costa,Mafra,Amaral,&Oliveira,2010).Although PCR is a rapid and sensitive technique,it is expensive and prone to generating false-positive signals (Baric,Kerschbamer,&Dalla,2006).Molecular marker methods such as random ampli fied polymorphic DNA (RAPD),simple sequence repeat (SSR),and ampli fied fragment length polymorphism (AFLP)techniques have been developed and optimized.DNA marker methods require that the samples are derived from a single cultivar,but also rely on the accessibility of*Corresponding authors.Tel./fax:þ861068981191.E-mail addresses:sulanb@ (S.Bai),yhe@ (Y.He).1Equal contribution to thiswork.Contents lists available at ScienceDirectFood Controljournal homep age:/locate/foodcont0956-7135/$e see front matter Ó2011Elsevier Ltd.All rights reserved.doi:10.1016/j.foodcont.2011.03.019Food Control 22(2011)1624e 1628sufficient reference cultivars.Evidently,these two limitations are important disadvantages(Wu et al.,2008).Microarray analysis has been used to simultaneously identify a large number of target DNA fragments on one chip.However,it requires expensive equipment and highly trained researchers to perform,thus limiting its general usage for routine sample identi-fication.In this study,we optimized a method for the detection of eight plant species-specific genes on the surface of optical thin-film biosensor chips(Zhong et al.,2003).Eight different vegetable oils (peanut,cotton,maize,sesame,sunflower,soybean,palm and rape oil)were detected.The advantage of this technology is that due to the optical characteristics of the thin-film biosensor chip surface, the experimental results can be visualized by unaided human eyes (Bai et al.,2007).Furthermore,this technique does not need a large and expensive instrumentation for data scanning,and may be adapted to any laboratory with a basic molecular biology facility. This technique has demonstrated to be a rapid,economical,highly sensitive and specific method for the detection of various crop components in vegetable oil.2.Materials and methods2.1.Preparation of samplesSeeds of peanut,cotton,palm,sesame,maize,sunflower, soybean and rape were purchased from the Chinese Academy of Agricultural Sciences.After7days growth,the leaves were collected and stored atÀ20 C for DNA extraction.The eight cor-responding oils were purchased from the market as controls for method validation.2.2.DNA extractionPlant genomic DNA extraction from leaves was conducted as previously described(Guillemaut&Drouard,1992).Briefly,plant tissue(100mg)was ground in liquid nitrogen.The powder was transferred into a1.5mL microcentrifuge tube and suspended in 700m L of CTAB buffer(1.4M NaCl,2%CTAB(w/v),100mM Tris, 15mM EDTA,pH8.0).The sample was incubated at65 C for30min with occasional gentle shaking and then centrifuged at12,000rpm for5min at room temperature.The supernatant(200m L)was decanted and mixed with200m L chloroform/isoamyl alcohol (24:1).After centrifugation(12,000rpm)for5min,the supernatant was mixed with1mL absolute ethanol and cooled atÀ20 C for 30min,and then centrifuged for1min.The residue was washed with75%ethanol and dried.The genomic DNA in the tube was dissolved in100m L of TE buffer(10mM Tris,1mM EDTA).The quality of the extractions was analyzed on1.5%agarose gels and the productivity was measured using a NanoDrop spectrophotometer (NanoDrop Technologies,Wilmington,DE).DNA samples were diluted to100ng/m L for further evaulation.Extraction of DNA from oils was conducted according to the protocol(unpublished)by Yin Chen at the Institute of Food Safety,Chinese Academy of Inspection and Quarantine.2.3.PCR amplificationPCR reactions were performed on an ABI2720Thermal Cycler (Applied Biosystems).PCR reaction mixtures(25m L each)were prepared by mixing1ÂPCR buffer,MgCl2(2.5mmol/L),dNTP (0.8mmol/L),primers(0.4m mol/L each;Table1),DNA templates (50ng)and rTaq(0.75U;TaKaRa,Japan).Amplifications were executed as follows:heating at94 C for5min for pre-denaturation, 36cycles of sequential heating at94 C for30s,57 C for30s,and72 C for30s,followed by afinal incubation at72 C for10min.The PCR products were analyzed by electrophoresis on2%agarose gels.Elec-trophoresis images were photographed using a GelDocXR system (Bio-Rad).2.4.Primer and probe synthesisOligonucleotides were designed against species genes of crop components in vegetable oil.These genes were verified species-specific genes based on the references shown in Table1.TheTable1Information about the PCR primers and probes used in this study.Species Target genes Sequences PCR fragment size ReferenceCanola Accg8F a:50-GAGAATGAGGAGGACCAAGCTCR b:50-bioTin-GGCGCAGCATCGGCTCTTP c:50-ALD d-aaaaaaaaaaGACGAACACCTATTAGACATTCGTTCCATTGGTCGATGGA160Hernández et al.,2001Cotton Sad1F:50-CCAAAGGAGGTGCCTGTTCAR:50-bioTin-TTGCTCATGAAATCCATCAP:50-ALD-aaaaaaaaaaGATTGAGATCTTTAAATCTTTGGAGGGCTGGGCTGAGAAC107Yang et al.,2005Maize Ivr1F:50-CCGCTGTATCACAAGGGCTGGTACCR:50-bioTin-TGTAGAGCATGACGATCCP:50-ALD-aaaaaaaaaaaCACTGGCTGCACCTACCGCTGGCCATGGTGCCCGATCACC247Hernández et al.,2004Soybean Lectin F:50-GCCCTCTACTCCACCCCCATCCR:50-bioTin-GCCCATCTGCAAGCCTTTTTGTGP:50-ALD-aaaaaaaaaaCATTTGGGACAAAGAAACCGGTAGCGTTGCCAGCTTCGCC118Bai et al.,2007Peanuts Arah2F:50-GCTCGAGAGGCCGAACCTR:50-bioTin-CAGCTCATTGCAACACCTP:50-ALD-aaaaaaaaaaGAGCAACATCTCATGCAGAAGATCCAACGTGACGAGGA 177Hird,Lloyd,Goodier,Brown,&Reece,2003Sunflower Helianthinin F:50-CTCGAGCACCTCCGGCTR:50-bioTin-GCCCTGCAAGGTTTGCTATCP:50-ALD-aaaaaaaaaaAGAGCCAATGAACAAGGAAGCAGGTGGGTGTCTTTC289Hernández et al.,2005Sesame Ses i1F:50-CACAGCAGGTTTACCAGAGGR:50-bioTin-TTATACATTTCCTCGCACAACCP:50-ALD-aaaaaaaaaaAACATGCGACCCCAGCAATGCCAATTCCGAGTTATCTT143Schöringhumer and Markl,2007Palm MT3-B F:50-TCCAAGGAGTTGTACGTTTTGTGR:50-bioTin-CGCAGTTAGAGCCGCATTTP:50-ALD-aaaaaaaaaaAATCATTTGTTGCAGCTACTTCGAGGAAGTCGTTGAGG109Zhang et al.,2009a Forward PCR primer.b Reverse PCR primer.c Probe.d ALD,aldehyde modification.S.Bai et al./Food Control22(2011)1624e16281625sequences of PCR primers and probes for the detection of the plant speci fic genes are also listed in Table 1.The genes selected were the invertase (Ivr1)gene of maize,the acetyl-CoA carboxylase (Accg8)gene of rape,the stearoyl-ACP desaturase (Sad1)gene of cotton,the Lectin gene of soybean,the Arachis hypogaea allergen II (Arah2)gene of peanuts,the Helianthinin gene of sun flower,the Sesi1gene for coding Sesamum indicum 2S albumin of sesame and the MT3-B gene of palm.The 50end of the reverse primers for each gene has a biotin modi fication.The 50end of the probes has an aldehyde modi fication and followed by ten deoxyadenosine residues (10dA).Oligonucle-otides were synthesized by Invitrogen company (Shanghai,China).2.5.Preparation of the optical thin-film biosensor chipsThe optical biosensor chip can transduce speci fic molecular interactions into visual signals,because after enzymatic catalysis the molecular deposits on the chip surface would change the wavelength of light re flected by the optical layer and thus generate a perceived color change.In order to provide an optical layer,the surface of the chips was coated with a 475Ålayer of silicon nitride (Si 3N 4;Bai et al.,2007,2010;Ostroff,Hopkins,Haeberli,Baouchi,&Polisky,1999;Zhong et al.,2003).To facilitate covalent attachment of biomolecules,the chip surface was functionalized with hydra-zine by coating with poly (Phe e Lys)for future covalent linkage of the capture probes (Jenison,Yang,Haeberli,&Polisky,2001).2.6.Standard assay procedureThe biosensor chips were loaded with the aldehyde-labeled probes by spotting (40nL per spot)a 1.0m M probe solution (in 0.1M sodium phosphate buffer,pH 7.8)via a robotic pipetting device (Biodot AD3200).The chips were allowed to sit at room temperature and 70%humidity for 2h,and then washed with 0.1%SDS.The PCR amplicon targets were then hybridized with probes on the chip.The PCR products (w 100fmol)were denatured at 95 C for 5min and hybridized on the chip for 10min at 50 C in 90m L of reaction buffer [5Âstandard saline citrate (SSC),5mg/mL acid-treated caseine (ATC)].After two washes with 0.1ÂSSC,the chips were incubated with an anti-biotin IgG-horseradish peroxidase (HRP)conjugate (Jackson Immuno Research;4:1000dilution from a 1mg/mL stock in a buffer containing 5ÂSSC:5mg/mL,ATC:10%glycerol)for 10min in hybridization buffer.After two washes in 0.1ÂSSC,100m L of tetramethylbenzidine (TMB,BioFx Laboratories,Owings Mills,MD)was added and incubated for 10min at room temperature.The chips were then rinsed in double-distilled H 2O,brie fly air-dried and visually examined by eyes.The chips can also be imaged with a dissection microscope fitted with a digital camera.3.Results and discussion3.1.Strategy for speci fic genes detection on thin-film biosensor chip This speci fic gene detection method for identi fication of plant species-speci fic genes in oil relies on the hybridization of bio-tinylated PCR fragments with probes covalently attached to a thin-film silicon biosensor chip in a speci fic array.In addition,one primer pair and one probe were synthesized for each gene.The reverse primer carries biotin at the 50end for subsequent detection (Table 1).The probes were labeled with aldehyde for covalent attachment to chip surface.The probes also had 10dA as spacers,followed by 40bp nucleotides corresponding to the sequence between the forward and reverse primers of the targets sequence.The hybridization reactions were completed after incubation for 10min,when probes complementarily combine with the target amplicons.After two washes with 1ÂSSC to remove mismatched molecules deposited,the immobilized biotinylated PCR fragment were incubated with anti-biotin IgG-HRP conjugate and precipi-table HRP substrate.The molecules deposited on the chip surface would cause a distinguishable color change from gold to blue,which could be detected by eyes or photographed by a simple digital-imaging system.3.2.De fining sensitivity and speci ficity of thin-film biosensor chips In order to determine the appropriate probe concentration for spotting on the chip,we randomly chose two probes for the rape Accg 8gene and cotton Sad 1gene.The probes were diluted to0.001,Fig.1.The design and results for speci ficity and sensitivity of plant species-speci fic genes detection on thin-film biosensor chips.Capturing probes (200nL)of the Accg 8and Sad 1genes were each spotted by hand at concentrations of 0.001,0.01,0.1,and 1m M,respectively (left panel).Biotin-dA20(M)was spotted as positive control.PCR amplicons of Sad 1(0,0.1,1,10,and 100fmol)in 100m L of reaction solution were hybridized on five identical chips (rightpanel).Fig.2.PCR ampli fication of target DNA fragments from eight vegetable nes1:M,DNA marker,DL 2000;1,lectin (Soybean);2,Helianthinin (Sun flower);3,Ses i 1(Sesame);4,MT3-B (Palm);5,Accg 8(Rape);6,Arah 2(Peanuts);7,Ivr 1(Maize);8,Sad 1(Cotton).S.Bai et al./Food Control 22(2011)1624e 162816260.01,0.1and 1m M,and 200nL of each serial diluted probe solution was manually spotted on the biosensor chip surface (Fig.1left panel).The chips were hybridized with Sad 1gene PCR products at concentrations of 0,0.1,1,10and 100fmol in 100m L total reaction volume.As expected,only the spots containing Sad 1gene probe were detected (Fig.1right panel),indicating the speci ficity of this assay.The results showed that the signal intensity decreased as the concentrations of probe and target DNA were reduced.However,0.1fmol of target can still be detected for the probe spotted at high concentration (1m M).Probe concentration higher than 1m M did not increase detection sensitivity at the arrangement of target DNA concentrations tested (data not shown).For economic consider-ations,probe concentration of 1m M was chosen for subsequent spotting.Of course,the other probes may have their own potential optimization on the sensitivity.3.3.Ampli fication of eight target gene fragments by PCRThe PCR products for the eight species are shown in Fig.2.The sizes of ampli fied fragments were the same as predicted (Table 1).Sequencing results con firmed that the target fragments were correct.The results showed that the speci ficity of the PCR ampli-fication of the target genes was acceptable.Target fragments were the main bright bands.Nonspeci fic ampli fication bands were very weak and should not affect chip hybridization.3.4.Chip design and detection of target genes using optical thin-film biosensor chipsAs shown in Fig.3(upper panel),the chips spotted by robotic pipetting (40nL per spot)were used to detect plant species-speci fic genes in oils.M refers to the positive control biotin-dA20that always exhibited a signal if the chip detection system worked.In this detection system,all other genes were negative controls for the target gene.The results showed that speci fic signals were detected for plant species-speci fic genes (Fig.3).The hybridization with cotton Sad1gene resulted in one set of colored dots (Fig.3,lower panel 8).The other seven gene targets showed their own speci fic sets of colored dots (Fig.3.1e 7).No false-positive signals were observed.To con firm this assay for the detection of real vegetable oils,we extracted the DNA from the corresponding eight vegetable oils and obtained the same results (data not show).4.ConclusionsWe have demonstrated that this optical thin-film biosensor chips detection method can speci fically detect trace amount of DNA as low as 0.1fmol,which is much more sensitive than previously reported (Doveri &Lee,2007).Moreover,the relatively simple equipment requirements make this method easily adaptable to other laboratories (Jenison et al.,2001).This technique has proven to be a rapid,simple,speci fic and sensitive method suitable fortheFig.3.Vegetable oil detection on a chip coated with capture probes spotted by a computer-controlled dispenser.The upper panel was the design for plant species-speci fic genes detection on a chip.Each spot comprised 40nL of 1m M capture probes solution spotted by a computer-controlled dispenser.Capture probes were spotted in the following order.M:biotin-dA20(positive control marker);1:lectin (Soybean);2:Helianthinin (Sun flower);3:Ses i 1(Sesame);4:MT3-B (Palm);5:Accg 8(Rape);6:Arah 2(Peanuts);7:Ivr 1(Maize);8:Sad 1(Cotton).The lower panel shows the results for detection of the eight species-speci fic genes on thin-film biosensor chips in 100m L of reaction solution.The PCR target amplicons (100fmol)from genes 1to 8were applied on identical chips.S.Bai et al./Food Control 22(2011)1624e 16281627detection of vegetable oils.Furthermore,the method offers simple visible detection by unaided human eyes,which makes it ideal for authentication of many other organisms.AcknowledgmentsThis work was supported by Beijing Municipal Education Commission(KM201110028010and KM200800005004),Beijing Municipal Natural Science Foundation(5112006and5082003), National Natural Science Foundation of China(30771094),Ministry of Agriculture of China(2009ZX08005-006B),and National Key Project of Foundation Research in China(2007CB948201)to He. 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