Research ArticleRapid and Accurate Identification of Adulterantsvia an Electronic Nose and DNA Identification PlatformIdentification of Fake Velvet Antlers as an Example

Guojie Xu Chunsheng Liu Caili Liao Xiaolei RenXinyue Zhang Xiaorui Fu and Xueyong Wang

School of Chinese Materia Medica Beijing University of Chinese Medicine No 6 Wang Jing Zhong Huan South StreetChaoyang District Beijing 100102 China

Correspondence should be addressed to Chunsheng Liu max liucs263net and Xueyong Wang wxyphd163com

Received 19 June 2015 Revised 26 October 2015 Accepted 27 October 2015

Academic Editor Kourosh Kalantar-Zadeh

Copyright copy 2016 Guojie Xu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

BackgroundAdulterants inChinesemedicines had always affected the efficacy of Chinesemedicines and resulted in safety problemsin drug use We aimed to take the identification of fake velvet antlers as an example for establishment of a rapid and accurateidentification platform for modern pharmaceutical companies and markets of Chinese medicine Methods In this study wedeveloped a novel electronic nose and DNA identification platform for identifying fake velvet antlers Electronic nose was designedfor rapid screening while accurate identification based on DNA method was performed when electronic nose fails to accuratelyidentify some complex samples Results Rapid identification of different velvet antlers based on electronic nose showed the goodidentification power while accurate identification based on DNA sequencing technology demonstrated a correct identification rateof 100 We also mined thirty conserved specific sites of fake velvet antlers which was useful for further work Conclusions Theresults suggested that electronic nose and DNA identification platform was effective for rapid and accurate identification of fakevelvet antlers It highly improved efficiency and decreased cost of identification and could provide a new sight and inspiration foridentification in the field of traditional Chinese medicine (TCM)

1 Introduction

Adulterants inChinesemedicines had always affected the effi-cacy of Chinese medicines and resulted in safety problems indrug use However modern pharmaceutical companies andmarkets of Chinese medicine do not have a suitable platformfor rapid and accurate identification of adulterants Velvetantler (Lu-Rong in China) for instance the nonossific hornof Cervus nippon or Cervus elaphus is a precious animal-sourced Chinese medicine in China Pharmacological stud-ies had proven its functions of anti-bone resorption [1]antiarthritis [2] and promoting chondrocyte proliferation[3] Unfortunately official velvet antlers are always replacedby the fake velvet antler ofRangifer tarandusdue to high profitin Chinese market [4ndash6] It is therefore necessary to developa rapid and accurate identification platform for identificationof adulterants

DNA sequencing technology which can mirror abundantDNA information is good candidate for accurate identifica-tion of adulterants [4ndash10] DNA sequences can be directlydetermined by fluorescence sensors of DNA sequencerHowever it is also always of low efficiency and high costElectronic nose is an intelligent design of simulating humanrsquosolfactory system Its array of metal oxide sensors interactingwith a broad range of chemicals with varying intensities isused to rapidly describe the substances of odors Comparedwith conventional DNA identification electronic nose hashigh efficiency and low cost for identification

It can be imaged that DNA sequencing method com-bined with electronic nose can highly improve efficiency anddecrease cost of identificationHere we introduced electronicnose into the discrimination of traditional Chinesemedicines(TCM) and further developed a novel electronic nose andDNA identification platform for identification of fake velvet

Hindawi Publishing CorporationJournal of SensorsVolume 2016 Article ID 1830843 7 pageshttpdxdoiorg10115520161830843

2 Journal of Sensors

This work

Samples

Grinding

Rapid screeningby electronic nose

Unclear samples

Determination of DNA sequenceby DNA sequencer

Accurate identification

Further work

WearableComplete database

DNA extractionNon-PCRNonsequencing

Figure 1 Instruction of electronic nose and DNA identificationplatform

Table 1 Details of the samples

Velvetantlers Source Assession number of DNA sequences in

Genbank

Authentic

C nippon

Sequences of current studyKJ205534ndashKJ205542Sequences of previous studyEU835711ndashEU835715GQ329010ndashGQ329015JF700148ndashJF700150

C elaphus

Sequences of current studyKJ205543ndashKJ205557Sequences of previous studyEU835707ndashEU835709JF443209ndashJF443224 JF700147GQ329007ndashGQ329009

Fake R tarandus

Sequences of current studyKJ205565ndashKJ205580Sequences of previous studyJF443384ndashJF443512

antlers (Figure 1) We aimed to take the identification of fakevelvet antlers as an example for establishment of a rapid andaccurate identification platform for modern pharmaceuticalcompanies and markets of Chinese medicine which is ofgreat significance to standardization normalization andmedication safety of Chinese medicine

2 Material and Methods

21 ExperimentalMaterials The samples were collected fromdifferent medicinal velvet antlers markets in China identifiedby Professor Chen Daixian of Dalian Institute for DrugControl according to the features of the pharmacopoeia ofChina and previous work [11 12] All the samples weredeposited into the specimen room of Beijing University ofChinese Medicine Details of the samples were listed inTable 1

22 Electronic Nose A FOX-3000 (Alpha MOS ToulouseFrance) which consisted of a sampling apparatus array of

Table 2 The components and main application of sensors of theFOX-3000 electronic nose

Number Name Main applicationS1 LY2LG Oxidizing gasS2 LY2G Ammonia carbon monoxideS3 LY2AA EthanolS4 LY2GH Ammoniaorganic amineS5 LY2gCTL Hydrogen sulfideS6 LY2gCT PropanebutaneS7 T301 Organic solventsS8 P101 HydrocarbonsS9 P102 MethaneS10 P102 FluorineS11 T702 Aromatic compoundsS12 PA2 Ethanol ammoniaorganic amine

sensors HS-100 autosampler air generator equipment andsoftware (Alpha Soft V11) for data recording and analyzingwas used The sensor array was composed of 12 metal oxidesensors divided into three chambers T P and LYThesemetaloxide sensors could catch the electrical signals from redoxreactionwith gaseous chemical compoundsThe componentsand main application of sensors of the FOX-3000 electronicnose were listed in Table 2 The response values of samplesof fake velvet antlers were recorded according to the previouswork [13] The data sets of official or authentic velvet antlersalso referred to our previous work [13]

23 Statistical Processing of Electronic Nose Each sample wasrepeated three times 10 response values at a period of time(5 s 10 s 15 s 20 s 25 s 30 s 35 s 40 s 45 s and 50 s) ofeach sensor were collected Raw response values of electronicnose were primary-processed artificially Some values withno response to all samples were deleted The resulting datasets of samples were analyzed by SPSS 190 based on lineardiscriminant analysis (LDA)

24 DNA Extraction and PCR Amplification Partial cyto-chrome oxidase subunit I (COI) gene of 618 bp was used fordiscrimination of different velvet antlers DNA was extractedusing a common animal DNA extraction kit (BiologicalTechnology Co Ltd China) The primers for amplificationof partial COI genes were CF (51015840-CGGTACTCTGTA-TCTACTATTTGGTG-31015840) and CR (51015840-GGGTGACCAAAG-AATCAGAATAAGTG-31015840) PCR was performed in a 50 120583Lreaction volume containing 10x PCR Buffer (50 120583L) dNTPs(04mM each) MgCl

2(30mM) CF (02 120583M) CR (02 120583M)

template DNA (240 ng) and Taq polymerase (025U) Theamplification profile consisted of 5min at 94∘C 35 cycles of30 s at 95∘C 30 s at 50∘C 60 s at 72∘C and an additional exten-sion for 10min at 72∘C The sequences of the PCR productswere determined by 3730xl DNAAnalyzer (Shanghai SangonBiological Technology Co Ltd China)

25 DNA Analysis Eighty DNA sequences of forty sampleswere determined by DNA Analyzer All the sequences were

Journal of Sensors 3

C nipponC elaphus

R tarandus

Function 1

minus50 minus25 00 25 50

minus50

minus25

00

25

50

Func

tion2

Figure 2 Data analysis of electronic nose based on LDA model

spliced by free ContigExpress software Sequences were thenaligned by clustalW throughMEGA 50 Specific sites for fakevelvet antlers were determined at last and arranged by Excelsoftware Phylogenetic trees were constructed by Neighbor-Joining methods using Kimurarsquos 2-parameter model withbootstrap testing of 1000 replicates through MEGA 50[14] 166 related sequences previously registered in Genbankdatabase (httpwwwncbinlmnihgov) were collected forfurther verifying discriminative power of COI sequences andconservation of specific sites The analysis method was thesame as procedure mentioned above

3 Results and Discussion

31 Rapid Discrimination Based on Electronic Nose Velvetantlers are animal-sourced medicinal materials Actuallyrare studies had reported about discrimination of animal-sourced medicinal materials based on specific smells Wefirstly established the electronic nose to assess the qualityof different deer antler slices [13] and this study is the firstattempt to test the electronic nose for discrimination of fakeanimal-sourced medicinal species

We previously had understood that LDA model wassuitable for modeling of velvet antlers [13] so LDA directlywas used to test their applicability of discriminating fakevelvet antlers in this study As a result most samples weresuccessfully discriminated based on LDA model (Figure 2)It could be implied that electronic nose based on LDAmodelwas effective for rapid identification of velvet antlers Resultsalso indicated that good response values to velvet antlers werecollected from S6 to S12 (data not shown) which were usedas important distinguishing sensors for fake velvet antlers

However we also found that a few samples which werehighlighted by red arrows in Figure 2 were not well discrim-inated To explore the reasons that affect the discriminationpower of electronic nose we classified the samples into fourpresentations including wax slices powder slices sand slicesand bone slices because we deduced that distinctive smellsfromdifferent presentationsmay cover up the species-specificsmells Results showed that smells of sand slices and boneslices were distinguishable but a few samples of wax slicesand powder slices were undistinguishable (Figure 3) Thisindicated that smells of wax slices and powder slices mayaffect the species-specific smells of samples

Traditionally identification of Chinese medicines alwaysdepends on appearance and microscopic features of samplesin enterprises and markets of Chinese medicine These kindsof traditional methods are of high subjectivity and alwaysrely on experience of identifier In comparison to othermethods electronic nose is more simple and objective Butsome problems of electronic nose should also be addressedin the future First the use of electronic nose in the field oftraditional Chinese medicine (TCM) is still in the beginningstages the establishment of big electronic nose database ofdifferent Chinese medicines is necessary for rapid identi-fication Second Chinese medicines are always processedby various methods thus the effects of different processingmethods on smells should be taken into considerationThirdin order to meet the need of identifiers proper identificationmodel should be built and portable electronic nose driveshould be developed for specific Chinese medicines Lastbut not least other methods such as DNA identificationmethod should be in federated manner applied to assurethe identification power when some other smells confuseddetection of electronic nose

32 Accurate Identification Based onDNASequencing PartialCOI gene about 600 bp had been acknowledged as uni-versal DNA identification sequence for discrimination ofanimal species [4] Velvet antlers are parts of animal bodiesTherefore it is feasible to use partial COI sequences fordiscrimination of different velvet antlers in this study

Specific sites of sequences of fake velvet antlers werescreened byMEGA 50Thirty conserved specific sites of fakevelvet antlers were finally mined in total including site 3site 21 site 27 site 45 site 48 site 54 site 78 site 81 site156 site 198 site 237 site 238 site 285 site 300 site 304 site330 site 385 site 393 site 429 site 435 site 444 site 457 site465 site 471 site 474 site 478 site 493 site 507 site 552 andsite 570 (Figure 4) These sites were also proven by the 166sequences fromGenbank databaseThe results suggested thatthese specific sites were useful for identifying the fake velvetantlers

Figure 5 shows the cluster analysis of velvet antlers basedon partial COI sequences As shown all the individuals ofR tarandus were obviously clustered together in one cladeso were 166 related sequences collected in Genbank databasewhich suggested that all the individuals of R tarandus werecompletely discriminated correctly The results proved thatpartial COI sequences showed the excellent identificationpower of velvet antlers with a correct identification rate of

4 Journal of Sensors

C nipponC elaphus

R tarandus

40 2minus2minus4

Function 1

minus4

minus2

0

2

4

Func

tion2

(a)

C nipponC elaphus

R tarandus

minus10

minus5

0

5

10

Func

tion2

minus5 0 10minus10 5

Function 1

(b)

C nipponC elaphus

R tarandus

minus20

minus10

0

10

20

Func

tion2

minus20 10 20minus10 0

Function 1

(c)

C nipponC elaphus

R tarandus

minus3

minus2

minus1

0

1

2

3

Func

tion2

0minus2 1 2minus1 3minus3

Function 1

(d)

Figure 3 Data analysis of velvet antlers of different presentations based on LDA model (a) Wax slices (b) Powder slices (c) Sand slices (d)Bone slices

100 Thus we determined DNA identification could be thegood candidate for accurate identification

DNA sequencing technology had been generally appliedin the field of TCM in recent years [4 15] it does havehigh accuracy but it is also of high cost and is time-consuming So newmethods in the future should be proposedto lower the cost and improve the efficiency We thought

that DNA specific amplification methods based on specificsites may come to our aid such as allele-specific PCR [15]and amplification refractory mutation system [16] Thesemethods can detect specific DNA sites without sequencingthus lowering the cost and improving the efficiency Andthe better one we believed is loop-mediated isothermalamplification [17] which can rapidly detect samples in a few

Journal of Sensors 5

R tarandus A C T G C C C T A AG C T T A C T A A T C C G T G C T G A A CT G G G C C A A C C T G G G A C C [48]C elaphus A T C G T C T T [48]C nippon A T C G T C T T [48]

R tarandus C T A C T C G G A GA C G A T C A A A T T T A T A A T G T A AT T G T A A C C G C A C A T G C A [96]C elaphus T T C T C

[96]C nippon G T T T C

[96]

R tarandus T T C G T A A T A AT T T T C T T T A T A G T A A T G C C A AT T A T A A T T G G A G G A T T T [144]C elaphus T [144]C nippon T A C [144]

R tarandus G G T A A T T G A CT T G T C C C T C T A A T A A T T G G T GC C C C A G A T A T A G C A T T C [192]C elaphus A T C C C T [192]C nippon A T C C [192]

R tarandus C C T C G G A T A AA T A A T A T A A G C T T C T G A C T T CT C C C T C C T T C T T T T C T A [240]C elaphus A C T C C T [240]C nippon A C T C C T [240]

R tarandus C T T C T T C T A GC A T C A T C C A T A A T T G A A G C T GG A G C A G G A A C A G G T T G A [288]

C elaphus A C T G C C [288]C nippon A T T G C C [288]

R tarandus A C T G T T T A C CC T C C T T T A G C T G G T A A C C T A GC T C A C G C A G G A G C T T C A [336]C elaphus A T C C C T T G [336]C nippon A T C C C T G [336]

R tarandus G T A G A C T T A AC T A T T T T C T C T T T A C A C T T A GC A G G T G T C T C C T C A A T T [384]C elaphus C G T C [384]C nippon C G C T G [384]

R tarandus T T A G G G G C A AT T A A C T T T A T T A C A A C A A T T AT T A A T A T A A A A C C T C C T [432]C elaphus C C C C [432]C nippon C C C C [432]

R tarandus G C T A T A T C A CA G T A T C A A A C C C C T T T A T T T GT A T G A T C T G T C T T A A T C [480]C elaphus C A T C G C A G [480]C nippon C A C C G C A G [480]

R tarandus A C T G C T G T A TT A T T A C T T C T C T C A C T T C C T GT A C T A G C A G C C G G A A T T [528]C elaphus C C C [528]C nippon C C C C [528]

R tarandus A C A A T A C T A TT A A C A G A C C G A A A T T T A A A T AC A A C T T T C T T C G A C C C A [576]C elaphus C C T [576]C nippon C C C T T [576]

R tarandus G C A G G A G G C GG G G A T C C C A T C C T A T A T C A A CA T T T A T T C T G A [618]C elaphus A T T C G [618]C nippon A T T C G [618]

Figure 4 Arrangement of all specific sites of fake velvet antlers

minutes without PCR amplifier and will play an importantrole in the further work Another nut we have to be facedwith is the DNA extraction of Chinese medicine As we allknow lots of secondarymetabolites are inChinesemedicinesdevelopment of novelDNAextraction and purificationmeth-ods for Chinese medicines will be important in the future

33 Application and Perspective of the Electronic Nose andDNA Identification Platform It had been reported that thetraditional authenticationmethods based on themorphology[12] andmicroscopic character [11 18] had been firstly appliedto discrimination of velvet antlers but these methods areassociated with high subjectivity Development of chemical

6 Journal of Sensors

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nippon

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandus

54

1519

65

24

33

10

100

8965

63

36

68

322952

5980

86

47

6951

89

66

001(a)

Cervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Rangifer tarandus

89

231298

96

73

39297

91

74

77

64

81

3121

71

37

2150

54

100

001(b)

Figure 5 Cluster analysis ofDNAsequences (a)DNAsequences frommaterials of present study (b)DNAsequences fromGenbankdatabase

method based on high performance capillary electrophoresis(HPCE) [19] then had improved the objectivity and accuracynevertheless it is always not satisfied in terms of practicalityand identification power In this study electronic nose andDNA identification platform integrating electronic nose andDNA sequencing technology provides a more flexible andpractical identification platform Electronic nose is designedfor rapid screening while accurate identification basedon DNA method is performed when electronic nose failsto accurately identify some complex samples which doesincrease the efficiency of identification and vivify the use(Figure 1)

In fact many traditional Chinese medicines are aromaticsuch as herbal medicines from Umbelliferae and Labiataeand electronic nose is exactly suitable for rapid identification

of these materials Meanwhile DNA identification methodwhich is recognized as an accurate identification methoduniversally can make up for defects of electronic nose in caseof unclear identification

Electronic nose and DNA identification platform devel-oped in this study will also be improved according to thevision mentioned above in our further study We believe itwill play an important role in the field of TCM

4 Conclusions

In this study rapid identification of different velvet antlersbased on electronic nose showed the good identificationpower while accurate identification based on DNA sequenc-ing technology showed a correct identification rate of 100

Journal of Sensors 7

The results further proved that electronic nose and DNAidentification platform covering the advantages of electronicnose andDNA sequencing technology was effective for rapidand accurate identification and highly improved efficiencyand decreased cost of identification It is also believed thatelectronic nose and DNA identification platform will beof great significance to standardization normalization andmedication safety of Chinese medicine

Abbreviations

TCM Traditional Chinese medicineHPCE High performance capillary electrophoresisLDA Linear discriminant analysisCOI Cytochrome oxidase subunit I

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Xueyong Wang and Chunsheng Liu conceived and designedthe study Guojie Xu and Caili Liao accomplished the wholeexperiment and wrote the paper Xinyue Zhang Xiaorui Fuand Xiaolei Ren accounted for data analysis collecting sam-ples and processing samples All authors read and approvedthe final version of the paper

Acknowledgments

The authors thank Huihui Duan of Beijing University ofChinese Medicine for supporting the study This work wassupported by Beijing Natural Science Foundation of China(7152094 and 7142101) and National Natural Science Founda-tion of China (81274011)

References

[1] K-H Kim K-S Kim B-J Choi et al ldquoAnti-bone resorptionactivity of deer antler aqua-acupunture the pilose antler ofCervus koreanTEMMINCK var mantchuricus Swinhoe (Noky-ong) in adjuvant-induced arthritic ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 497ndash506 2005

[2] K-S Kim Y-H Choi K-H Kim et al ldquoProtective andanti-arthritic effects of deer antler aqua-acupuncture (DAA)inhibiting dihydroorotate dehydrogenase on phosphate ions-mediated chondrocyte apoptosis and rat collagen-inducedarthritisrdquo International Immunopharmacology vol 4 no 7 pp963ndash973 2004

[3] J-H Lin L-X Deng Z-Y Wu L Chen and L Zhang ldquoPiloseantler polypeptides promote chondrocyte proliferation via thetyrosine kinase signaling pathwayrdquo Journal of OccupationalMedicine and Toxicology vol 6 no 1 article 27 2011

[4] D Yan J Y Luo YM Han et al ldquoForensic DNA barcoding andbio-response studies of animal horn products used in tradi-tional medicinerdquo PLoS ONE vol 8 no 2 Article ID e558542013

[5] X Wang C Liu R Zhang L Huang and G Cui ldquoEstablish-ment of allele-specific diagnostic PCRmethod for identificationof zantlersrdquoZhongguo Zhongyao Zazhi vol 34 no 23 pp 3013ndash3016 2009

[6] R Zhang C-S Liu L-Q Huang X-Y Wang G-H Cui andL Dong ldquoStudy on the identification of Cornu Cervi Pan-totrichum with DNA barcodingrdquo Chinese Pharmaceutical Jour-nal vol 46 no 4 pp 263ndash266 2011

[7] M Natonek-Wioeniewska E Słota and B Kalisz ldquoUse ofcytochrome b polymorphism for species identification of bio-logical material derived from cattle sheep goats roe deer andred deerrdquo Folia Biologica vol 58 no 1-2 pp 47ndash50 2010

[8] Q Wang X Zhang H-Y Zhang et al ldquoIdentification of 12animal species meat by T-RFLP on the 12S rRNA generdquo MeatScience vol 85 no 2 pp 265ndash269 2010

[9] G D Haynes and E K Latch ldquoIdentification of novel singlenucleotide polymorphisms (SNPs) in deer (Odocoileus spp)using the BovineSNP50 Beadchiprdquo PLoS ONE vol 7 no 5Article ID e36536 2012

[10] Y H Kim E S Kim B S Ko et al ldquoA PCR-based assay fordiscriminating Cervus and Rangifer (Cervidae) antlers withmitochondrial DNA polymorphismsrdquo Journal of Animal Sci-ence vol 90 no 7 pp 2075ndash2083 2012

[11] L Liu and T-G Kang ldquoMicroscopical identification and hier-archical cluster analysis of seven kinds of pilose antler velvetrdquoZhong Yao Cai vol 32 no 3 pp 345ndash347 2009

[12] D Chen Y Guo and W Ren ldquoCharacter identification of 12kinds of pilose antler medicinal materialsrdquo Zhong Yao Cai vol22 no 9 pp 441ndash444 1999

[13] G Xu C Liao X Ren et al ldquoRapid assessment of qualityof deer antler slices by using an electronic nose coupled withchemometric analysisrdquoBrazilian Journal of Pharmacognosy vol24 no 6 pp 716ndash721 2014

[14] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[15] G Xu X Wang C Liu et al ldquoAuthentication of official Da-huang by sequencing and multiplex allele-specific PCR of ashortmaturaseK generdquoGenome vol 56 no 2 pp 109ndash113 2013

[16] XWangG Xu C Liu et al ldquoDevelopment of deft amplificationrefractory mutation sequencing system (ARMSS) for discrimi-nating Pilos antler based on a short cytochrome b (Cytb) generdquoMitochondrial DNA 2014

[17] K Hsieh B S Ferguson M Eisenstein K W Plaxco and HT Soh ldquoIntegrated electrochemical microsystems for geneticdetection of pathogens at the point of carerdquo Accounts ofChemical Research vol 48 no 4 pp 911ndash920 2015

[18] D J Ye ldquoMicroscopic identification of pilose antler and deer-hornrdquo Zhong Yao Tong Bao vol 11 no 12 pp 17ndash19 1986

[19] Z Yan R Y Yuan C Y Wang Z H Zhang L P Cai and J KWang ldquoStudy on fingerprint of Corn cervi pantotrichum byHPCErdquo Food Science and Technology vol 34 no 4 pp 254ndash2582009

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DistributedSensor Networks

International Journal of

2 Journal of Sensors

This work

Samples

Grinding

Rapid screeningby electronic nose

Unclear samples

Determination of DNA sequenceby DNA sequencer

Accurate identification

Further work

WearableComplete database

DNA extractionNon-PCRNonsequencing

Figure 1 Instruction of electronic nose and DNA identificationplatform

Table 1 Details of the samples

Velvetantlers Source Assession number of DNA sequences in

Genbank

Authentic

C nippon

Sequences of current studyKJ205534ndashKJ205542Sequences of previous studyEU835711ndashEU835715GQ329010ndashGQ329015JF700148ndashJF700150

C elaphus

Sequences of current studyKJ205543ndashKJ205557Sequences of previous studyEU835707ndashEU835709JF443209ndashJF443224 JF700147GQ329007ndashGQ329009

Fake R tarandus

Sequences of current studyKJ205565ndashKJ205580Sequences of previous studyJF443384ndashJF443512

antlers (Figure 1) We aimed to take the identification of fakevelvet antlers as an example for establishment of a rapid andaccurate identification platform for modern pharmaceuticalcompanies and markets of Chinese medicine which is ofgreat significance to standardization normalization andmedication safety of Chinese medicine

2 Material and Methods

21 ExperimentalMaterials The samples were collected fromdifferent medicinal velvet antlers markets in China identifiedby Professor Chen Daixian of Dalian Institute for DrugControl according to the features of the pharmacopoeia ofChina and previous work [11 12] All the samples weredeposited into the specimen room of Beijing University ofChinese Medicine Details of the samples were listed inTable 1

22 Electronic Nose A FOX-3000 (Alpha MOS ToulouseFrance) which consisted of a sampling apparatus array of

Table 2 The components and main application of sensors of theFOX-3000 electronic nose

Number Name Main applicationS1 LY2LG Oxidizing gasS2 LY2G Ammonia carbon monoxideS3 LY2AA EthanolS4 LY2GH Ammoniaorganic amineS5 LY2gCTL Hydrogen sulfideS6 LY2gCT PropanebutaneS7 T301 Organic solventsS8 P101 HydrocarbonsS9 P102 MethaneS10 P102 FluorineS11 T702 Aromatic compoundsS12 PA2 Ethanol ammoniaorganic amine

sensors HS-100 autosampler air generator equipment andsoftware (Alpha Soft V11) for data recording and analyzingwas used The sensor array was composed of 12 metal oxidesensors divided into three chambers T P and LYThesemetaloxide sensors could catch the electrical signals from redoxreactionwith gaseous chemical compoundsThe componentsand main application of sensors of the FOX-3000 electronicnose were listed in Table 2 The response values of samplesof fake velvet antlers were recorded according to the previouswork [13] The data sets of official or authentic velvet antlersalso referred to our previous work [13]

23 Statistical Processing of Electronic Nose Each sample wasrepeated three times 10 response values at a period of time(5 s 10 s 15 s 20 s 25 s 30 s 35 s 40 s 45 s and 50 s) ofeach sensor were collected Raw response values of electronicnose were primary-processed artificially Some values withno response to all samples were deleted The resulting datasets of samples were analyzed by SPSS 190 based on lineardiscriminant analysis (LDA)

24 DNA Extraction and PCR Amplification Partial cyto-chrome oxidase subunit I (COI) gene of 618 bp was used fordiscrimination of different velvet antlers DNA was extractedusing a common animal DNA extraction kit (BiologicalTechnology Co Ltd China) The primers for amplificationof partial COI genes were CF (51015840-CGGTACTCTGTA-TCTACTATTTGGTG-31015840) and CR (51015840-GGGTGACCAAAG-AATCAGAATAAGTG-31015840) PCR was performed in a 50 120583Lreaction volume containing 10x PCR Buffer (50 120583L) dNTPs(04mM each) MgCl

2(30mM) CF (02 120583M) CR (02 120583M)

template DNA (240 ng) and Taq polymerase (025U) Theamplification profile consisted of 5min at 94∘C 35 cycles of30 s at 95∘C 30 s at 50∘C 60 s at 72∘C and an additional exten-sion for 10min at 72∘C The sequences of the PCR productswere determined by 3730xl DNAAnalyzer (Shanghai SangonBiological Technology Co Ltd China)

25 DNA Analysis Eighty DNA sequences of forty sampleswere determined by DNA Analyzer All the sequences were

Journal of Sensors 3

C nipponC elaphus

R tarandus

Function 1

minus50 minus25 00 25 50

minus50

minus25

00

25

50

Func

tion2

Figure 2 Data analysis of electronic nose based on LDA model

spliced by free ContigExpress software Sequences were thenaligned by clustalW throughMEGA 50 Specific sites for fakevelvet antlers were determined at last and arranged by Excelsoftware Phylogenetic trees were constructed by Neighbor-Joining methods using Kimurarsquos 2-parameter model withbootstrap testing of 1000 replicates through MEGA 50[14] 166 related sequences previously registered in Genbankdatabase (httpwwwncbinlmnihgov) were collected forfurther verifying discriminative power of COI sequences andconservation of specific sites The analysis method was thesame as procedure mentioned above

3 Results and Discussion

31 Rapid Discrimination Based on Electronic Nose Velvetantlers are animal-sourced medicinal materials Actuallyrare studies had reported about discrimination of animal-sourced medicinal materials based on specific smells Wefirstly established the electronic nose to assess the qualityof different deer antler slices [13] and this study is the firstattempt to test the electronic nose for discrimination of fakeanimal-sourced medicinal species

We previously had understood that LDA model wassuitable for modeling of velvet antlers [13] so LDA directlywas used to test their applicability of discriminating fakevelvet antlers in this study As a result most samples weresuccessfully discriminated based on LDA model (Figure 2)It could be implied that electronic nose based on LDAmodelwas effective for rapid identification of velvet antlers Resultsalso indicated that good response values to velvet antlers werecollected from S6 to S12 (data not shown) which were usedas important distinguishing sensors for fake velvet antlers

However we also found that a few samples which werehighlighted by red arrows in Figure 2 were not well discrim-inated To explore the reasons that affect the discriminationpower of electronic nose we classified the samples into fourpresentations including wax slices powder slices sand slicesand bone slices because we deduced that distinctive smellsfromdifferent presentationsmay cover up the species-specificsmells Results showed that smells of sand slices and boneslices were distinguishable but a few samples of wax slicesand powder slices were undistinguishable (Figure 3) Thisindicated that smells of wax slices and powder slices mayaffect the species-specific smells of samples

Traditionally identification of Chinese medicines alwaysdepends on appearance and microscopic features of samplesin enterprises and markets of Chinese medicine These kindsof traditional methods are of high subjectivity and alwaysrely on experience of identifier In comparison to othermethods electronic nose is more simple and objective Butsome problems of electronic nose should also be addressedin the future First the use of electronic nose in the field oftraditional Chinese medicine (TCM) is still in the beginningstages the establishment of big electronic nose database ofdifferent Chinese medicines is necessary for rapid identi-fication Second Chinese medicines are always processedby various methods thus the effects of different processingmethods on smells should be taken into considerationThirdin order to meet the need of identifiers proper identificationmodel should be built and portable electronic nose driveshould be developed for specific Chinese medicines Lastbut not least other methods such as DNA identificationmethod should be in federated manner applied to assurethe identification power when some other smells confuseddetection of electronic nose

32 Accurate Identification Based onDNASequencing PartialCOI gene about 600 bp had been acknowledged as uni-versal DNA identification sequence for discrimination ofanimal species [4] Velvet antlers are parts of animal bodiesTherefore it is feasible to use partial COI sequences fordiscrimination of different velvet antlers in this study

Specific sites of sequences of fake velvet antlers werescreened byMEGA 50Thirty conserved specific sites of fakevelvet antlers were finally mined in total including site 3site 21 site 27 site 45 site 48 site 54 site 78 site 81 site156 site 198 site 237 site 238 site 285 site 300 site 304 site330 site 385 site 393 site 429 site 435 site 444 site 457 site465 site 471 site 474 site 478 site 493 site 507 site 552 andsite 570 (Figure 4) These sites were also proven by the 166sequences fromGenbank databaseThe results suggested thatthese specific sites were useful for identifying the fake velvetantlers

Figure 5 shows the cluster analysis of velvet antlers basedon partial COI sequences As shown all the individuals ofR tarandus were obviously clustered together in one cladeso were 166 related sequences collected in Genbank databasewhich suggested that all the individuals of R tarandus werecompletely discriminated correctly The results proved thatpartial COI sequences showed the excellent identificationpower of velvet antlers with a correct identification rate of

4 Journal of Sensors

C nipponC elaphus

R tarandus

40 2minus2minus4

Function 1

minus4

minus2

0

2

4

Func

tion2

(a)

C nipponC elaphus

R tarandus

minus10

minus5

0

5

10

Func

tion2

minus5 0 10minus10 5

Function 1

(b)

C nipponC elaphus

R tarandus

minus20

minus10

0

10

20

Func

tion2

minus20 10 20minus10 0

Function 1

(c)

C nipponC elaphus

R tarandus

minus3

minus2

minus1

0

1

2

3

Func

tion2

0minus2 1 2minus1 3minus3

Function 1

(d)

Figure 3 Data analysis of velvet antlers of different presentations based on LDA model (a) Wax slices (b) Powder slices (c) Sand slices (d)Bone slices

100 Thus we determined DNA identification could be thegood candidate for accurate identification

DNA sequencing technology had been generally appliedin the field of TCM in recent years [4 15] it does havehigh accuracy but it is also of high cost and is time-consuming So newmethods in the future should be proposedto lower the cost and improve the efficiency We thought

that DNA specific amplification methods based on specificsites may come to our aid such as allele-specific PCR [15]and amplification refractory mutation system [16] Thesemethods can detect specific DNA sites without sequencingthus lowering the cost and improving the efficiency Andthe better one we believed is loop-mediated isothermalamplification [17] which can rapidly detect samples in a few

Journal of Sensors 5

R tarandus A C T G C C C T A AG C T T A C T A A T C C G T G C T G A A CT G G G C C A A C C T G G G A C C [48]C elaphus A T C G T C T T [48]C nippon A T C G T C T T [48]

R tarandus C T A C T C G G A GA C G A T C A A A T T T A T A A T G T A AT T G T A A C C G C A C A T G C A [96]C elaphus T T C T C

[96]C nippon G T T T C

[96]

R tarandus T T C G T A A T A AT T T T C T T T A T A G T A A T G C C A AT T A T A A T T G G A G G A T T T [144]C elaphus T [144]C nippon T A C [144]

R tarandus G G T A A T T G A CT T G T C C C T C T A A T A A T T G G T GC C C C A G A T A T A G C A T T C [192]C elaphus A T C C C T [192]C nippon A T C C [192]

R tarandus C C T C G G A T A AA T A A T A T A A G C T T C T G A C T T CT C C C T C C T T C T T T T C T A [240]C elaphus A C T C C T [240]C nippon A C T C C T [240]

R tarandus C T T C T T C T A GC A T C A T C C A T A A T T G A A G C T GG A G C A G G A A C A G G T T G A [288]

C elaphus A C T G C C [288]C nippon A T T G C C [288]

R tarandus A C T G T T T A C CC T C C T T T A G C T G G T A A C C T A GC T C A C G C A G G A G C T T C A [336]C elaphus A T C C C T T G [336]C nippon A T C C C T G [336]

R tarandus G T A G A C T T A AC T A T T T T C T C T T T A C A C T T A GC A G G T G T C T C C T C A A T T [384]C elaphus C G T C [384]C nippon C G C T G [384]

R tarandus T T A G G G G C A AT T A A C T T T A T T A C A A C A A T T AT T A A T A T A A A A C C T C C T [432]C elaphus C C C C [432]C nippon C C C C [432]

R tarandus G C T A T A T C A CA G T A T C A A A C C C C T T T A T T T GT A T G A T C T G T C T T A A T C [480]C elaphus C A T C G C A G [480]C nippon C A C C G C A G [480]

R tarandus A C T G C T G T A TT A T T A C T T C T C T C A C T T C C T GT A C T A G C A G C C G G A A T T [528]C elaphus C C C [528]C nippon C C C C [528]

R tarandus A C A A T A C T A TT A A C A G A C C G A A A T T T A A A T AC A A C T T T C T T C G A C C C A [576]C elaphus C C T [576]C nippon C C C T T [576]

R tarandus G C A G G A G G C GG G G A T C C C A T C C T A T A T C A A CA T T T A T T C T G A [618]C elaphus A T T C G [618]C nippon A T T C G [618]

Figure 4 Arrangement of all specific sites of fake velvet antlers

minutes without PCR amplifier and will play an importantrole in the further work Another nut we have to be facedwith is the DNA extraction of Chinese medicine As we allknow lots of secondarymetabolites are inChinesemedicinesdevelopment of novelDNAextraction and purificationmeth-ods for Chinese medicines will be important in the future

33 Application and Perspective of the Electronic Nose andDNA Identification Platform It had been reported that thetraditional authenticationmethods based on themorphology[12] andmicroscopic character [11 18] had been firstly appliedto discrimination of velvet antlers but these methods areassociated with high subjectivity Development of chemical

6 Journal of Sensors

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nippon

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandus

54

1519

65

24

33

10

100

8965

63

36

68

322952

5980

86

47

6951

89

66

001(a)

Cervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Rangifer tarandus

89

231298

96

73

39297

91

74

77

64

81

3121

71

37

2150

54

100

001(b)

Figure 5 Cluster analysis ofDNAsequences (a)DNAsequences frommaterials of present study (b)DNAsequences fromGenbankdatabase

method based on high performance capillary electrophoresis(HPCE) [19] then had improved the objectivity and accuracynevertheless it is always not satisfied in terms of practicalityand identification power In this study electronic nose andDNA identification platform integrating electronic nose andDNA sequencing technology provides a more flexible andpractical identification platform Electronic nose is designedfor rapid screening while accurate identification basedon DNA method is performed when electronic nose failsto accurately identify some complex samples which doesincrease the efficiency of identification and vivify the use(Figure 1)

In fact many traditional Chinese medicines are aromaticsuch as herbal medicines from Umbelliferae and Labiataeand electronic nose is exactly suitable for rapid identification

of these materials Meanwhile DNA identification methodwhich is recognized as an accurate identification methoduniversally can make up for defects of electronic nose in caseof unclear identification

Electronic nose and DNA identification platform devel-oped in this study will also be improved according to thevision mentioned above in our further study We believe itwill play an important role in the field of TCM

4 Conclusions

In this study rapid identification of different velvet antlersbased on electronic nose showed the good identificationpower while accurate identification based on DNA sequenc-ing technology showed a correct identification rate of 100

Journal of Sensors 7

The results further proved that electronic nose and DNAidentification platform covering the advantages of electronicnose andDNA sequencing technology was effective for rapidand accurate identification and highly improved efficiencyand decreased cost of identification It is also believed thatelectronic nose and DNA identification platform will beof great significance to standardization normalization andmedication safety of Chinese medicine

Abbreviations

TCM Traditional Chinese medicineHPCE High performance capillary electrophoresisLDA Linear discriminant analysisCOI Cytochrome oxidase subunit I

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Xueyong Wang and Chunsheng Liu conceived and designedthe study Guojie Xu and Caili Liao accomplished the wholeexperiment and wrote the paper Xinyue Zhang Xiaorui Fuand Xiaolei Ren accounted for data analysis collecting sam-ples and processing samples All authors read and approvedthe final version of the paper

Acknowledgments

The authors thank Huihui Duan of Beijing University ofChinese Medicine for supporting the study This work wassupported by Beijing Natural Science Foundation of China(7152094 and 7142101) and National Natural Science Founda-tion of China (81274011)

References

[1] K-H Kim K-S Kim B-J Choi et al ldquoAnti-bone resorptionactivity of deer antler aqua-acupunture the pilose antler ofCervus koreanTEMMINCK var mantchuricus Swinhoe (Noky-ong) in adjuvant-induced arthritic ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 497ndash506 2005

[2] K-S Kim Y-H Choi K-H Kim et al ldquoProtective andanti-arthritic effects of deer antler aqua-acupuncture (DAA)inhibiting dihydroorotate dehydrogenase on phosphate ions-mediated chondrocyte apoptosis and rat collagen-inducedarthritisrdquo International Immunopharmacology vol 4 no 7 pp963ndash973 2004

[3] J-H Lin L-X Deng Z-Y Wu L Chen and L Zhang ldquoPiloseantler polypeptides promote chondrocyte proliferation via thetyrosine kinase signaling pathwayrdquo Journal of OccupationalMedicine and Toxicology vol 6 no 1 article 27 2011

[4] D Yan J Y Luo YM Han et al ldquoForensic DNA barcoding andbio-response studies of animal horn products used in tradi-tional medicinerdquo PLoS ONE vol 8 no 2 Article ID e558542013

[5] X Wang C Liu R Zhang L Huang and G Cui ldquoEstablish-ment of allele-specific diagnostic PCRmethod for identificationof zantlersrdquoZhongguo Zhongyao Zazhi vol 34 no 23 pp 3013ndash3016 2009

[6] R Zhang C-S Liu L-Q Huang X-Y Wang G-H Cui andL Dong ldquoStudy on the identification of Cornu Cervi Pan-totrichum with DNA barcodingrdquo Chinese Pharmaceutical Jour-nal vol 46 no 4 pp 263ndash266 2011

[7] M Natonek-Wioeniewska E Słota and B Kalisz ldquoUse ofcytochrome b polymorphism for species identification of bio-logical material derived from cattle sheep goats roe deer andred deerrdquo Folia Biologica vol 58 no 1-2 pp 47ndash50 2010

[8] Q Wang X Zhang H-Y Zhang et al ldquoIdentification of 12animal species meat by T-RFLP on the 12S rRNA generdquo MeatScience vol 85 no 2 pp 265ndash269 2010

[9] G D Haynes and E K Latch ldquoIdentification of novel singlenucleotide polymorphisms (SNPs) in deer (Odocoileus spp)using the BovineSNP50 Beadchiprdquo PLoS ONE vol 7 no 5Article ID e36536 2012

[10] Y H Kim E S Kim B S Ko et al ldquoA PCR-based assay fordiscriminating Cervus and Rangifer (Cervidae) antlers withmitochondrial DNA polymorphismsrdquo Journal of Animal Sci-ence vol 90 no 7 pp 2075ndash2083 2012

[11] L Liu and T-G Kang ldquoMicroscopical identification and hier-archical cluster analysis of seven kinds of pilose antler velvetrdquoZhong Yao Cai vol 32 no 3 pp 345ndash347 2009

[12] D Chen Y Guo and W Ren ldquoCharacter identification of 12kinds of pilose antler medicinal materialsrdquo Zhong Yao Cai vol22 no 9 pp 441ndash444 1999

[13] G Xu C Liao X Ren et al ldquoRapid assessment of qualityof deer antler slices by using an electronic nose coupled withchemometric analysisrdquoBrazilian Journal of Pharmacognosy vol24 no 6 pp 716ndash721 2014

[14] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[15] G Xu X Wang C Liu et al ldquoAuthentication of official Da-huang by sequencing and multiplex allele-specific PCR of ashortmaturaseK generdquoGenome vol 56 no 2 pp 109ndash113 2013

[16] XWangG Xu C Liu et al ldquoDevelopment of deft amplificationrefractory mutation sequencing system (ARMSS) for discrimi-nating Pilos antler based on a short cytochrome b (Cytb) generdquoMitochondrial DNA 2014

[17] K Hsieh B S Ferguson M Eisenstein K W Plaxco and HT Soh ldquoIntegrated electrochemical microsystems for geneticdetection of pathogens at the point of carerdquo Accounts ofChemical Research vol 48 no 4 pp 911ndash920 2015

[18] D J Ye ldquoMicroscopic identification of pilose antler and deer-hornrdquo Zhong Yao Tong Bao vol 11 no 12 pp 17ndash19 1986

[19] Z Yan R Y Yuan C Y Wang Z H Zhang L P Cai and J KWang ldquoStudy on fingerprint of Corn cervi pantotrichum byHPCErdquo Food Science and Technology vol 34 no 4 pp 254ndash2582009

International Journal of

AerospaceEngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

RoboticsJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Active and Passive Electronic Components

Control Scienceand Engineering

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

RotatingMachinery

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

Journal ofEngineeringVolume 2014

Submit your manuscripts athttpwwwhindawicom

VLSI Design

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Shock and Vibration

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Civil EngineeringAdvances in

Acoustics and VibrationAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Electrical and Computer Engineering

Journal of

Advances inOptoElectronics

Hindawi Publishing Corporation httpwwwhindawicom

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

SensorsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Modelling amp Simulation in EngineeringHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chemical EngineeringInternational Journal of Antennas and

Propagation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Navigation and Observation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

DistributedSensor Networks

International Journal of

Journal of Sensors 3

C nipponC elaphus

R tarandus

Function 1

minus50 minus25 00 25 50

minus50

minus25

00

25

50

Func

tion2

Figure 2 Data analysis of electronic nose based on LDA model

spliced by free ContigExpress software Sequences were thenaligned by clustalW throughMEGA 50 Specific sites for fakevelvet antlers were determined at last and arranged by Excelsoftware Phylogenetic trees were constructed by Neighbor-Joining methods using Kimurarsquos 2-parameter model withbootstrap testing of 1000 replicates through MEGA 50[14] 166 related sequences previously registered in Genbankdatabase (httpwwwncbinlmnihgov) were collected forfurther verifying discriminative power of COI sequences andconservation of specific sites The analysis method was thesame as procedure mentioned above

3 Results and Discussion

31 Rapid Discrimination Based on Electronic Nose Velvetantlers are animal-sourced medicinal materials Actuallyrare studies had reported about discrimination of animal-sourced medicinal materials based on specific smells Wefirstly established the electronic nose to assess the qualityof different deer antler slices [13] and this study is the firstattempt to test the electronic nose for discrimination of fakeanimal-sourced medicinal species

We previously had understood that LDA model wassuitable for modeling of velvet antlers [13] so LDA directlywas used to test their applicability of discriminating fakevelvet antlers in this study As a result most samples weresuccessfully discriminated based on LDA model (Figure 2)It could be implied that electronic nose based on LDAmodelwas effective for rapid identification of velvet antlers Resultsalso indicated that good response values to velvet antlers werecollected from S6 to S12 (data not shown) which were usedas important distinguishing sensors for fake velvet antlers

However we also found that a few samples which werehighlighted by red arrows in Figure 2 were not well discrim-inated To explore the reasons that affect the discriminationpower of electronic nose we classified the samples into fourpresentations including wax slices powder slices sand slicesand bone slices because we deduced that distinctive smellsfromdifferent presentationsmay cover up the species-specificsmells Results showed that smells of sand slices and boneslices were distinguishable but a few samples of wax slicesand powder slices were undistinguishable (Figure 3) Thisindicated that smells of wax slices and powder slices mayaffect the species-specific smells of samples

Traditionally identification of Chinese medicines alwaysdepends on appearance and microscopic features of samplesin enterprises and markets of Chinese medicine These kindsof traditional methods are of high subjectivity and alwaysrely on experience of identifier In comparison to othermethods electronic nose is more simple and objective Butsome problems of electronic nose should also be addressedin the future First the use of electronic nose in the field oftraditional Chinese medicine (TCM) is still in the beginningstages the establishment of big electronic nose database ofdifferent Chinese medicines is necessary for rapid identi-fication Second Chinese medicines are always processedby various methods thus the effects of different processingmethods on smells should be taken into considerationThirdin order to meet the need of identifiers proper identificationmodel should be built and portable electronic nose driveshould be developed for specific Chinese medicines Lastbut not least other methods such as DNA identificationmethod should be in federated manner applied to assurethe identification power when some other smells confuseddetection of electronic nose

32 Accurate Identification Based onDNASequencing PartialCOI gene about 600 bp had been acknowledged as uni-versal DNA identification sequence for discrimination ofanimal species [4] Velvet antlers are parts of animal bodiesTherefore it is feasible to use partial COI sequences fordiscrimination of different velvet antlers in this study

Specific sites of sequences of fake velvet antlers werescreened byMEGA 50Thirty conserved specific sites of fakevelvet antlers were finally mined in total including site 3site 21 site 27 site 45 site 48 site 54 site 78 site 81 site156 site 198 site 237 site 238 site 285 site 300 site 304 site330 site 385 site 393 site 429 site 435 site 444 site 457 site465 site 471 site 474 site 478 site 493 site 507 site 552 andsite 570 (Figure 4) These sites were also proven by the 166sequences fromGenbank databaseThe results suggested thatthese specific sites were useful for identifying the fake velvetantlers

Figure 5 shows the cluster analysis of velvet antlers basedon partial COI sequences As shown all the individuals ofR tarandus were obviously clustered together in one cladeso were 166 related sequences collected in Genbank databasewhich suggested that all the individuals of R tarandus werecompletely discriminated correctly The results proved thatpartial COI sequences showed the excellent identificationpower of velvet antlers with a correct identification rate of

4 Journal of Sensors

C nipponC elaphus

R tarandus

40 2minus2minus4

Function 1

minus4

minus2

0

2

4

Func

tion2

(a)

C nipponC elaphus

R tarandus

minus10

minus5

0

5

10

Func

tion2

minus5 0 10minus10 5

Function 1

(b)

C nipponC elaphus

R tarandus

minus20

minus10

0

10

20

Func

tion2

minus20 10 20minus10 0

Function 1

(c)

C nipponC elaphus

R tarandus

minus3

minus2

minus1

0

1

2

3

Func

tion2

0minus2 1 2minus1 3minus3

Function 1

(d)

Figure 3 Data analysis of velvet antlers of different presentations based on LDA model (a) Wax slices (b) Powder slices (c) Sand slices (d)Bone slices

100 Thus we determined DNA identification could be thegood candidate for accurate identification

DNA sequencing technology had been generally appliedin the field of TCM in recent years [4 15] it does havehigh accuracy but it is also of high cost and is time-consuming So newmethods in the future should be proposedto lower the cost and improve the efficiency We thought

that DNA specific amplification methods based on specificsites may come to our aid such as allele-specific PCR [15]and amplification refractory mutation system [16] Thesemethods can detect specific DNA sites without sequencingthus lowering the cost and improving the efficiency Andthe better one we believed is loop-mediated isothermalamplification [17] which can rapidly detect samples in a few

Journal of Sensors 5

R tarandus A C T G C C C T A AG C T T A C T A A T C C G T G C T G A A CT G G G C C A A C C T G G G A C C [48]C elaphus A T C G T C T T [48]C nippon A T C G T C T T [48]

R tarandus C T A C T C G G A GA C G A T C A A A T T T A T A A T G T A AT T G T A A C C G C A C A T G C A [96]C elaphus T T C T C

[96]C nippon G T T T C

[96]

R tarandus T T C G T A A T A AT T T T C T T T A T A G T A A T G C C A AT T A T A A T T G G A G G A T T T [144]C elaphus T [144]C nippon T A C [144]

R tarandus G G T A A T T G A CT T G T C C C T C T A A T A A T T G G T GC C C C A G A T A T A G C A T T C [192]C elaphus A T C C C T [192]C nippon A T C C [192]

R tarandus C C T C G G A T A AA T A A T A T A A G C T T C T G A C T T CT C C C T C C T T C T T T T C T A [240]C elaphus A C T C C T [240]C nippon A C T C C T [240]

R tarandus C T T C T T C T A GC A T C A T C C A T A A T T G A A G C T GG A G C A G G A A C A G G T T G A [288]

C elaphus A C T G C C [288]C nippon A T T G C C [288]

R tarandus A C T G T T T A C CC T C C T T T A G C T G G T A A C C T A GC T C A C G C A G G A G C T T C A [336]C elaphus A T C C C T T G [336]C nippon A T C C C T G [336]

R tarandus G T A G A C T T A AC T A T T T T C T C T T T A C A C T T A GC A G G T G T C T C C T C A A T T [384]C elaphus C G T C [384]C nippon C G C T G [384]

R tarandus T T A G G G G C A AT T A A C T T T A T T A C A A C A A T T AT T A A T A T A A A A C C T C C T [432]C elaphus C C C C [432]C nippon C C C C [432]

R tarandus G C T A T A T C A CA G T A T C A A A C C C C T T T A T T T GT A T G A T C T G T C T T A A T C [480]C elaphus C A T C G C A G [480]C nippon C A C C G C A G [480]

R tarandus A C T G C T G T A TT A T T A C T T C T C T C A C T T C C T GT A C T A G C A G C C G G A A T T [528]C elaphus C C C [528]C nippon C C C C [528]

R tarandus A C A A T A C T A TT A A C A G A C C G A A A T T T A A A T AC A A C T T T C T T C G A C C C A [576]C elaphus C C T [576]C nippon C C C T T [576]

R tarandus G C A G G A G G C GG G G A T C C C A T C C T A T A T C A A CA T T T A T T C T G A [618]C elaphus A T T C G [618]C nippon A T T C G [618]

Figure 4 Arrangement of all specific sites of fake velvet antlers

minutes without PCR amplifier and will play an importantrole in the further work Another nut we have to be facedwith is the DNA extraction of Chinese medicine As we allknow lots of secondarymetabolites are inChinesemedicinesdevelopment of novelDNAextraction and purificationmeth-ods for Chinese medicines will be important in the future

33 Application and Perspective of the Electronic Nose andDNA Identification Platform It had been reported that thetraditional authenticationmethods based on themorphology[12] andmicroscopic character [11 18] had been firstly appliedto discrimination of velvet antlers but these methods areassociated with high subjectivity Development of chemical

6 Journal of Sensors

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nippon

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandus

54

1519

65

24

33

10

100

8965

63

36

68

322952

5980

86

47

6951

89

66

001(a)

Cervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Rangifer tarandus

89

231298

96

73

39297

91

74

77

64

81

3121

71

37

2150

54

100

001(b)

Figure 5 Cluster analysis ofDNAsequences (a)DNAsequences frommaterials of present study (b)DNAsequences fromGenbankdatabase

method based on high performance capillary electrophoresis(HPCE) [19] then had improved the objectivity and accuracynevertheless it is always not satisfied in terms of practicalityand identification power In this study electronic nose andDNA identification platform integrating electronic nose andDNA sequencing technology provides a more flexible andpractical identification platform Electronic nose is designedfor rapid screening while accurate identification basedon DNA method is performed when electronic nose failsto accurately identify some complex samples which doesincrease the efficiency of identification and vivify the use(Figure 1)

In fact many traditional Chinese medicines are aromaticsuch as herbal medicines from Umbelliferae and Labiataeand electronic nose is exactly suitable for rapid identification

of these materials Meanwhile DNA identification methodwhich is recognized as an accurate identification methoduniversally can make up for defects of electronic nose in caseof unclear identification

Electronic nose and DNA identification platform devel-oped in this study will also be improved according to thevision mentioned above in our further study We believe itwill play an important role in the field of TCM

4 Conclusions

In this study rapid identification of different velvet antlersbased on electronic nose showed the good identificationpower while accurate identification based on DNA sequenc-ing technology showed a correct identification rate of 100

Journal of Sensors 7

The results further proved that electronic nose and DNAidentification platform covering the advantages of electronicnose andDNA sequencing technology was effective for rapidand accurate identification and highly improved efficiencyand decreased cost of identification It is also believed thatelectronic nose and DNA identification platform will beof great significance to standardization normalization andmedication safety of Chinese medicine

Abbreviations

TCM Traditional Chinese medicineHPCE High performance capillary electrophoresisLDA Linear discriminant analysisCOI Cytochrome oxidase subunit I

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Xueyong Wang and Chunsheng Liu conceived and designedthe study Guojie Xu and Caili Liao accomplished the wholeexperiment and wrote the paper Xinyue Zhang Xiaorui Fuand Xiaolei Ren accounted for data analysis collecting sam-ples and processing samples All authors read and approvedthe final version of the paper

Acknowledgments

The authors thank Huihui Duan of Beijing University ofChinese Medicine for supporting the study This work wassupported by Beijing Natural Science Foundation of China(7152094 and 7142101) and National Natural Science Founda-tion of China (81274011)

References

[1] K-H Kim K-S Kim B-J Choi et al ldquoAnti-bone resorptionactivity of deer antler aqua-acupunture the pilose antler ofCervus koreanTEMMINCK var mantchuricus Swinhoe (Noky-ong) in adjuvant-induced arthritic ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 497ndash506 2005

[2] K-S Kim Y-H Choi K-H Kim et al ldquoProtective andanti-arthritic effects of deer antler aqua-acupuncture (DAA)inhibiting dihydroorotate dehydrogenase on phosphate ions-mediated chondrocyte apoptosis and rat collagen-inducedarthritisrdquo International Immunopharmacology vol 4 no 7 pp963ndash973 2004

[3] J-H Lin L-X Deng Z-Y Wu L Chen and L Zhang ldquoPiloseantler polypeptides promote chondrocyte proliferation via thetyrosine kinase signaling pathwayrdquo Journal of OccupationalMedicine and Toxicology vol 6 no 1 article 27 2011

[4] D Yan J Y Luo YM Han et al ldquoForensic DNA barcoding andbio-response studies of animal horn products used in tradi-tional medicinerdquo PLoS ONE vol 8 no 2 Article ID e558542013

[5] X Wang C Liu R Zhang L Huang and G Cui ldquoEstablish-ment of allele-specific diagnostic PCRmethod for identificationof zantlersrdquoZhongguo Zhongyao Zazhi vol 34 no 23 pp 3013ndash3016 2009

[6] R Zhang C-S Liu L-Q Huang X-Y Wang G-H Cui andL Dong ldquoStudy on the identification of Cornu Cervi Pan-totrichum with DNA barcodingrdquo Chinese Pharmaceutical Jour-nal vol 46 no 4 pp 263ndash266 2011

[7] M Natonek-Wioeniewska E Słota and B Kalisz ldquoUse ofcytochrome b polymorphism for species identification of bio-logical material derived from cattle sheep goats roe deer andred deerrdquo Folia Biologica vol 58 no 1-2 pp 47ndash50 2010

[8] Q Wang X Zhang H-Y Zhang et al ldquoIdentification of 12animal species meat by T-RFLP on the 12S rRNA generdquo MeatScience vol 85 no 2 pp 265ndash269 2010

[9] G D Haynes and E K Latch ldquoIdentification of novel singlenucleotide polymorphisms (SNPs) in deer (Odocoileus spp)using the BovineSNP50 Beadchiprdquo PLoS ONE vol 7 no 5Article ID e36536 2012

[10] Y H Kim E S Kim B S Ko et al ldquoA PCR-based assay fordiscriminating Cervus and Rangifer (Cervidae) antlers withmitochondrial DNA polymorphismsrdquo Journal of Animal Sci-ence vol 90 no 7 pp 2075ndash2083 2012

[11] L Liu and T-G Kang ldquoMicroscopical identification and hier-archical cluster analysis of seven kinds of pilose antler velvetrdquoZhong Yao Cai vol 32 no 3 pp 345ndash347 2009

[12] D Chen Y Guo and W Ren ldquoCharacter identification of 12kinds of pilose antler medicinal materialsrdquo Zhong Yao Cai vol22 no 9 pp 441ndash444 1999

[13] G Xu C Liao X Ren et al ldquoRapid assessment of qualityof deer antler slices by using an electronic nose coupled withchemometric analysisrdquoBrazilian Journal of Pharmacognosy vol24 no 6 pp 716ndash721 2014

[14] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[15] G Xu X Wang C Liu et al ldquoAuthentication of official Da-huang by sequencing and multiplex allele-specific PCR of ashortmaturaseK generdquoGenome vol 56 no 2 pp 109ndash113 2013

[16] XWangG Xu C Liu et al ldquoDevelopment of deft amplificationrefractory mutation sequencing system (ARMSS) for discrimi-nating Pilos antler based on a short cytochrome b (Cytb) generdquoMitochondrial DNA 2014

[17] K Hsieh B S Ferguson M Eisenstein K W Plaxco and HT Soh ldquoIntegrated electrochemical microsystems for geneticdetection of pathogens at the point of carerdquo Accounts ofChemical Research vol 48 no 4 pp 911ndash920 2015

[18] D J Ye ldquoMicroscopic identification of pilose antler and deer-hornrdquo Zhong Yao Tong Bao vol 11 no 12 pp 17ndash19 1986

[19] Z Yan R Y Yuan C Y Wang Z H Zhang L P Cai and J KWang ldquoStudy on fingerprint of Corn cervi pantotrichum byHPCErdquo Food Science and Technology vol 34 no 4 pp 254ndash2582009

International Journal of

AerospaceEngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

RoboticsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Active and Passive Electronic Components

Control Scienceand Engineering

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

RotatingMachinery

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

Journal ofEngineeringVolume 2014

Submit your manuscripts athttpwwwhindawicom

VLSI Design

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Shock and Vibration

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Civil EngineeringAdvances in

Acoustics and VibrationAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Electrical and Computer Engineering

Journal of

Advances inOptoElectronics

Hindawi Publishing Corporation httpwwwhindawicom

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

SensorsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Modelling amp Simulation in EngineeringHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chemical EngineeringInternational Journal of Antennas and

Propagation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Navigation and Observation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

DistributedSensor Networks

International Journal of

4 Journal of Sensors

C nipponC elaphus

R tarandus

40 2minus2minus4

Function 1

minus4

minus2

0

2

4

Func

tion2

(a)

C nipponC elaphus

R tarandus

minus10

minus5

0

5

10

Func

tion2

minus5 0 10minus10 5

Function 1

(b)

C nipponC elaphus

R tarandus

minus20

minus10

0

10

20

Func

tion2

minus20 10 20minus10 0

Function 1

(c)

C nipponC elaphus

R tarandus

minus3

minus2

minus1

0

1

2

3

Func

tion2

0minus2 1 2minus1 3minus3

Function 1

(d)

Figure 3 Data analysis of velvet antlers of different presentations based on LDA model (a) Wax slices (b) Powder slices (c) Sand slices (d)Bone slices

100 Thus we determined DNA identification could be thegood candidate for accurate identification

DNA sequencing technology had been generally appliedin the field of TCM in recent years [4 15] it does havehigh accuracy but it is also of high cost and is time-consuming So newmethods in the future should be proposedto lower the cost and improve the efficiency We thought

that DNA specific amplification methods based on specificsites may come to our aid such as allele-specific PCR [15]and amplification refractory mutation system [16] Thesemethods can detect specific DNA sites without sequencingthus lowering the cost and improving the efficiency Andthe better one we believed is loop-mediated isothermalamplification [17] which can rapidly detect samples in a few

Journal of Sensors 5

R tarandus A C T G C C C T A AG C T T A C T A A T C C G T G C T G A A CT G G G C C A A C C T G G G A C C [48]C elaphus A T C G T C T T [48]C nippon A T C G T C T T [48]

R tarandus C T A C T C G G A GA C G A T C A A A T T T A T A A T G T A AT T G T A A C C G C A C A T G C A [96]C elaphus T T C T C

[96]C nippon G T T T C

[96]

R tarandus T T C G T A A T A AT T T T C T T T A T A G T A A T G C C A AT T A T A A T T G G A G G A T T T [144]C elaphus T [144]C nippon T A C [144]

R tarandus G G T A A T T G A CT T G T C C C T C T A A T A A T T G G T GC C C C A G A T A T A G C A T T C [192]C elaphus A T C C C T [192]C nippon A T C C [192]

R tarandus C C T C G G A T A AA T A A T A T A A G C T T C T G A C T T CT C C C T C C T T C T T T T C T A [240]C elaphus A C T C C T [240]C nippon A C T C C T [240]

R tarandus C T T C T T C T A GC A T C A T C C A T A A T T G A A G C T GG A G C A G G A A C A G G T T G A [288]

C elaphus A C T G C C [288]C nippon A T T G C C [288]

R tarandus A C T G T T T A C CC T C C T T T A G C T G G T A A C C T A GC T C A C G C A G G A G C T T C A [336]C elaphus A T C C C T T G [336]C nippon A T C C C T G [336]

R tarandus G T A G A C T T A AC T A T T T T C T C T T T A C A C T T A GC A G G T G T C T C C T C A A T T [384]C elaphus C G T C [384]C nippon C G C T G [384]

R tarandus T T A G G G G C A AT T A A C T T T A T T A C A A C A A T T AT T A A T A T A A A A C C T C C T [432]C elaphus C C C C [432]C nippon C C C C [432]

R tarandus G C T A T A T C A CA G T A T C A A A C C C C T T T A T T T GT A T G A T C T G T C T T A A T C [480]C elaphus C A T C G C A G [480]C nippon C A C C G C A G [480]

R tarandus A C T G C T G T A TT A T T A C T T C T C T C A C T T C C T GT A C T A G C A G C C G G A A T T [528]C elaphus C C C [528]C nippon C C C C [528]

R tarandus A C A A T A C T A TT A A C A G A C C G A A A T T T A A A T AC A A C T T T C T T C G A C C C A [576]C elaphus C C T [576]C nippon C C C T T [576]

R tarandus G C A G G A G G C GG G G A T C C C A T C C T A T A T C A A CA T T T A T T C T G A [618]C elaphus A T T C G [618]C nippon A T T C G [618]

Figure 4 Arrangement of all specific sites of fake velvet antlers

minutes without PCR amplifier and will play an importantrole in the further work Another nut we have to be facedwith is the DNA extraction of Chinese medicine As we allknow lots of secondarymetabolites are inChinesemedicinesdevelopment of novelDNAextraction and purificationmeth-ods for Chinese medicines will be important in the future

33 Application and Perspective of the Electronic Nose andDNA Identification Platform It had been reported that thetraditional authenticationmethods based on themorphology[12] andmicroscopic character [11 18] had been firstly appliedto discrimination of velvet antlers but these methods areassociated with high subjectivity Development of chemical

6 Journal of Sensors

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nippon

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandus

54

1519

65

24

33

10

100

8965

63

36

68

322952

5980

86

47

6951

89

66

001(a)

Cervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Rangifer tarandus

89

231298

96

73

39297

91

74

77

64

81

3121

71

37

2150

54

100

001(b)

Figure 5 Cluster analysis ofDNAsequences (a)DNAsequences frommaterials of present study (b)DNAsequences fromGenbankdatabase

method based on high performance capillary electrophoresis(HPCE) [19] then had improved the objectivity and accuracynevertheless it is always not satisfied in terms of practicalityand identification power In this study electronic nose andDNA identification platform integrating electronic nose andDNA sequencing technology provides a more flexible andpractical identification platform Electronic nose is designedfor rapid screening while accurate identification basedon DNA method is performed when electronic nose failsto accurately identify some complex samples which doesincrease the efficiency of identification and vivify the use(Figure 1)

In fact many traditional Chinese medicines are aromaticsuch as herbal medicines from Umbelliferae and Labiataeand electronic nose is exactly suitable for rapid identification

of these materials Meanwhile DNA identification methodwhich is recognized as an accurate identification methoduniversally can make up for defects of electronic nose in caseof unclear identification

Electronic nose and DNA identification platform devel-oped in this study will also be improved according to thevision mentioned above in our further study We believe itwill play an important role in the field of TCM

4 Conclusions

In this study rapid identification of different velvet antlersbased on electronic nose showed the good identificationpower while accurate identification based on DNA sequenc-ing technology showed a correct identification rate of 100

Journal of Sensors 7

The results further proved that electronic nose and DNAidentification platform covering the advantages of electronicnose andDNA sequencing technology was effective for rapidand accurate identification and highly improved efficiencyand decreased cost of identification It is also believed thatelectronic nose and DNA identification platform will beof great significance to standardization normalization andmedication safety of Chinese medicine

Abbreviations

TCM Traditional Chinese medicineHPCE High performance capillary electrophoresisLDA Linear discriminant analysisCOI Cytochrome oxidase subunit I

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Xueyong Wang and Chunsheng Liu conceived and designedthe study Guojie Xu and Caili Liao accomplished the wholeexperiment and wrote the paper Xinyue Zhang Xiaorui Fuand Xiaolei Ren accounted for data analysis collecting sam-ples and processing samples All authors read and approvedthe final version of the paper

Acknowledgments

The authors thank Huihui Duan of Beijing University ofChinese Medicine for supporting the study This work wassupported by Beijing Natural Science Foundation of China(7152094 and 7142101) and National Natural Science Founda-tion of China (81274011)

References

[1] K-H Kim K-S Kim B-J Choi et al ldquoAnti-bone resorptionactivity of deer antler aqua-acupunture the pilose antler ofCervus koreanTEMMINCK var mantchuricus Swinhoe (Noky-ong) in adjuvant-induced arthritic ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 497ndash506 2005

[2] K-S Kim Y-H Choi K-H Kim et al ldquoProtective andanti-arthritic effects of deer antler aqua-acupuncture (DAA)inhibiting dihydroorotate dehydrogenase on phosphate ions-mediated chondrocyte apoptosis and rat collagen-inducedarthritisrdquo International Immunopharmacology vol 4 no 7 pp963ndash973 2004

[3] J-H Lin L-X Deng Z-Y Wu L Chen and L Zhang ldquoPiloseantler polypeptides promote chondrocyte proliferation via thetyrosine kinase signaling pathwayrdquo Journal of OccupationalMedicine and Toxicology vol 6 no 1 article 27 2011

[4] D Yan J Y Luo YM Han et al ldquoForensic DNA barcoding andbio-response studies of animal horn products used in tradi-tional medicinerdquo PLoS ONE vol 8 no 2 Article ID e558542013

[5] X Wang C Liu R Zhang L Huang and G Cui ldquoEstablish-ment of allele-specific diagnostic PCRmethod for identificationof zantlersrdquoZhongguo Zhongyao Zazhi vol 34 no 23 pp 3013ndash3016 2009

[6] R Zhang C-S Liu L-Q Huang X-Y Wang G-H Cui andL Dong ldquoStudy on the identification of Cornu Cervi Pan-totrichum with DNA barcodingrdquo Chinese Pharmaceutical Jour-nal vol 46 no 4 pp 263ndash266 2011

[7] M Natonek-Wioeniewska E Słota and B Kalisz ldquoUse ofcytochrome b polymorphism for species identification of bio-logical material derived from cattle sheep goats roe deer andred deerrdquo Folia Biologica vol 58 no 1-2 pp 47ndash50 2010

[8] Q Wang X Zhang H-Y Zhang et al ldquoIdentification of 12animal species meat by T-RFLP on the 12S rRNA generdquo MeatScience vol 85 no 2 pp 265ndash269 2010

[9] G D Haynes and E K Latch ldquoIdentification of novel singlenucleotide polymorphisms (SNPs) in deer (Odocoileus spp)using the BovineSNP50 Beadchiprdquo PLoS ONE vol 7 no 5Article ID e36536 2012

[10] Y H Kim E S Kim B S Ko et al ldquoA PCR-based assay fordiscriminating Cervus and Rangifer (Cervidae) antlers withmitochondrial DNA polymorphismsrdquo Journal of Animal Sci-ence vol 90 no 7 pp 2075ndash2083 2012

[11] L Liu and T-G Kang ldquoMicroscopical identification and hier-archical cluster analysis of seven kinds of pilose antler velvetrdquoZhong Yao Cai vol 32 no 3 pp 345ndash347 2009

[12] D Chen Y Guo and W Ren ldquoCharacter identification of 12kinds of pilose antler medicinal materialsrdquo Zhong Yao Cai vol22 no 9 pp 441ndash444 1999

[13] G Xu C Liao X Ren et al ldquoRapid assessment of qualityof deer antler slices by using an electronic nose coupled withchemometric analysisrdquoBrazilian Journal of Pharmacognosy vol24 no 6 pp 716ndash721 2014

[14] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[15] G Xu X Wang C Liu et al ldquoAuthentication of official Da-huang by sequencing and multiplex allele-specific PCR of ashortmaturaseK generdquoGenome vol 56 no 2 pp 109ndash113 2013

[16] XWangG Xu C Liu et al ldquoDevelopment of deft amplificationrefractory mutation sequencing system (ARMSS) for discrimi-nating Pilos antler based on a short cytochrome b (Cytb) generdquoMitochondrial DNA 2014

[17] K Hsieh B S Ferguson M Eisenstein K W Plaxco and HT Soh ldquoIntegrated electrochemical microsystems for geneticdetection of pathogens at the point of carerdquo Accounts ofChemical Research vol 48 no 4 pp 911ndash920 2015

[18] D J Ye ldquoMicroscopic identification of pilose antler and deer-hornrdquo Zhong Yao Tong Bao vol 11 no 12 pp 17ndash19 1986

[19] Z Yan R Y Yuan C Y Wang Z H Zhang L P Cai and J KWang ldquoStudy on fingerprint of Corn cervi pantotrichum byHPCErdquo Food Science and Technology vol 34 no 4 pp 254ndash2582009

International Journal of

AerospaceEngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

RoboticsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Active and Passive Electronic Components

Control Scienceand Engineering

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

RotatingMachinery

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

Journal ofEngineeringVolume 2014

Submit your manuscripts athttpwwwhindawicom

VLSI Design

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Shock and Vibration

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Civil EngineeringAdvances in

Acoustics and VibrationAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Electrical and Computer Engineering

Journal of

Advances inOptoElectronics

Hindawi Publishing Corporation httpwwwhindawicom

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

SensorsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Modelling amp Simulation in EngineeringHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chemical EngineeringInternational Journal of Antennas and

Propagation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Navigation and Observation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

DistributedSensor Networks

International Journal of

Journal of Sensors 5

R tarandus A C T G C C C T A AG C T T A C T A A T C C G T G C T G A A CT G G G C C A A C C T G G G A C C [48]C elaphus A T C G T C T T [48]C nippon A T C G T C T T [48]

R tarandus C T A C T C G G A GA C G A T C A A A T T T A T A A T G T A AT T G T A A C C G C A C A T G C A [96]C elaphus T T C T C

[96]C nippon G T T T C

[96]

R tarandus T T C G T A A T A AT T T T C T T T A T A G T A A T G C C A AT T A T A A T T G G A G G A T T T [144]C elaphus T [144]C nippon T A C [144]

R tarandus G G T A A T T G A CT T G T C C C T C T A A T A A T T G G T GC C C C A G A T A T A G C A T T C [192]C elaphus A T C C C T [192]C nippon A T C C [192]

R tarandus C C T C G G A T A AA T A A T A T A A G C T T C T G A C T T CT C C C T C C T T C T T T T C T A [240]C elaphus A C T C C T [240]C nippon A C T C C T [240]

R tarandus C T T C T T C T A GC A T C A T C C A T A A T T G A A G C T GG A G C A G G A A C A G G T T G A [288]

C elaphus A C T G C C [288]C nippon A T T G C C [288]

R tarandus A C T G T T T A C CC T C C T T T A G C T G G T A A C C T A GC T C A C G C A G G A G C T T C A [336]C elaphus A T C C C T T G [336]C nippon A T C C C T G [336]

R tarandus G T A G A C T T A AC T A T T T T C T C T T T A C A C T T A GC A G G T G T C T C C T C A A T T [384]C elaphus C G T C [384]C nippon C G C T G [384]

R tarandus T T A G G G G C A AT T A A C T T T A T T A C A A C A A T T AT T A A T A T A A A A C C T C C T [432]C elaphus C C C C [432]C nippon C C C C [432]

R tarandus G C T A T A T C A CA G T A T C A A A C C C C T T T A T T T GT A T G A T C T G T C T T A A T C [480]C elaphus C A T C G C A G [480]C nippon C A C C G C A G [480]

R tarandus A C T G C T G T A TT A T T A C T T C T C T C A C T T C C T GT A C T A G C A G C C G G A A T T [528]C elaphus C C C [528]C nippon C C C C [528]

R tarandus A C A A T A C T A TT A A C A G A C C G A A A T T T A A A T AC A A C T T T C T T C G A C C C A [576]C elaphus C C T [576]C nippon C C C T T [576]

R tarandus G C A G G A G G C GG G G A T C C C A T C C T A T A T C A A CA T T T A T T C T G A [618]C elaphus A T T C G [618]C nippon A T T C G [618]

Figure 4 Arrangement of all specific sites of fake velvet antlers

minutes without PCR amplifier and will play an importantrole in the further work Another nut we have to be facedwith is the DNA extraction of Chinese medicine As we allknow lots of secondarymetabolites are inChinesemedicinesdevelopment of novelDNAextraction and purificationmeth-ods for Chinese medicines will be important in the future

33 Application and Perspective of the Electronic Nose andDNA Identification Platform It had been reported that thetraditional authenticationmethods based on themorphology[12] andmicroscopic character [11 18] had been firstly appliedto discrimination of velvet antlers but these methods areassociated with high subjectivity Development of chemical

6 Journal of Sensors

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nippon

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandus

54

1519

65

24

33

10

100

8965

63

36

68

322952

5980

86

47

6951

89

66

001(a)

Cervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Rangifer tarandus

89

231298

96

73

39297

91

74

77

64

81

3121

71

37

2150

54

100

001(b)

Figure 5 Cluster analysis ofDNAsequences (a)DNAsequences frommaterials of present study (b)DNAsequences fromGenbankdatabase

method based on high performance capillary electrophoresis(HPCE) [19] then had improved the objectivity and accuracynevertheless it is always not satisfied in terms of practicalityand identification power In this study electronic nose andDNA identification platform integrating electronic nose andDNA sequencing technology provides a more flexible andpractical identification platform Electronic nose is designedfor rapid screening while accurate identification basedon DNA method is performed when electronic nose failsto accurately identify some complex samples which doesincrease the efficiency of identification and vivify the use(Figure 1)

In fact many traditional Chinese medicines are aromaticsuch as herbal medicines from Umbelliferae and Labiataeand electronic nose is exactly suitable for rapid identification

of these materials Meanwhile DNA identification methodwhich is recognized as an accurate identification methoduniversally can make up for defects of electronic nose in caseof unclear identification

Electronic nose and DNA identification platform devel-oped in this study will also be improved according to thevision mentioned above in our further study We believe itwill play an important role in the field of TCM

4 Conclusions

In this study rapid identification of different velvet antlersbased on electronic nose showed the good identificationpower while accurate identification based on DNA sequenc-ing technology showed a correct identification rate of 100

Journal of Sensors 7

The results further proved that electronic nose and DNAidentification platform covering the advantages of electronicnose andDNA sequencing technology was effective for rapidand accurate identification and highly improved efficiencyand decreased cost of identification It is also believed thatelectronic nose and DNA identification platform will beof great significance to standardization normalization andmedication safety of Chinese medicine

Abbreviations

TCM Traditional Chinese medicineHPCE High performance capillary electrophoresisLDA Linear discriminant analysisCOI Cytochrome oxidase subunit I

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Xueyong Wang and Chunsheng Liu conceived and designedthe study Guojie Xu and Caili Liao accomplished the wholeexperiment and wrote the paper Xinyue Zhang Xiaorui Fuand Xiaolei Ren accounted for data analysis collecting sam-ples and processing samples All authors read and approvedthe final version of the paper

Acknowledgments

The authors thank Huihui Duan of Beijing University ofChinese Medicine for supporting the study This work wassupported by Beijing Natural Science Foundation of China(7152094 and 7142101) and National Natural Science Founda-tion of China (81274011)

References

[1] K-H Kim K-S Kim B-J Choi et al ldquoAnti-bone resorptionactivity of deer antler aqua-acupunture the pilose antler ofCervus koreanTEMMINCK var mantchuricus Swinhoe (Noky-ong) in adjuvant-induced arthritic ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 497ndash506 2005

[2] K-S Kim Y-H Choi K-H Kim et al ldquoProtective andanti-arthritic effects of deer antler aqua-acupuncture (DAA)inhibiting dihydroorotate dehydrogenase on phosphate ions-mediated chondrocyte apoptosis and rat collagen-inducedarthritisrdquo International Immunopharmacology vol 4 no 7 pp963ndash973 2004

[3] J-H Lin L-X Deng Z-Y Wu L Chen and L Zhang ldquoPiloseantler polypeptides promote chondrocyte proliferation via thetyrosine kinase signaling pathwayrdquo Journal of OccupationalMedicine and Toxicology vol 6 no 1 article 27 2011

[4] D Yan J Y Luo YM Han et al ldquoForensic DNA barcoding andbio-response studies of animal horn products used in tradi-tional medicinerdquo PLoS ONE vol 8 no 2 Article ID e558542013

[5] X Wang C Liu R Zhang L Huang and G Cui ldquoEstablish-ment of allele-specific diagnostic PCRmethod for identificationof zantlersrdquoZhongguo Zhongyao Zazhi vol 34 no 23 pp 3013ndash3016 2009

[6] R Zhang C-S Liu L-Q Huang X-Y Wang G-H Cui andL Dong ldquoStudy on the identification of Cornu Cervi Pan-totrichum with DNA barcodingrdquo Chinese Pharmaceutical Jour-nal vol 46 no 4 pp 263ndash266 2011

[7] M Natonek-Wioeniewska E Słota and B Kalisz ldquoUse ofcytochrome b polymorphism for species identification of bio-logical material derived from cattle sheep goats roe deer andred deerrdquo Folia Biologica vol 58 no 1-2 pp 47ndash50 2010

[8] Q Wang X Zhang H-Y Zhang et al ldquoIdentification of 12animal species meat by T-RFLP on the 12S rRNA generdquo MeatScience vol 85 no 2 pp 265ndash269 2010

[9] G D Haynes and E K Latch ldquoIdentification of novel singlenucleotide polymorphisms (SNPs) in deer (Odocoileus spp)using the BovineSNP50 Beadchiprdquo PLoS ONE vol 7 no 5Article ID e36536 2012

[10] Y H Kim E S Kim B S Ko et al ldquoA PCR-based assay fordiscriminating Cervus and Rangifer (Cervidae) antlers withmitochondrial DNA polymorphismsrdquo Journal of Animal Sci-ence vol 90 no 7 pp 2075ndash2083 2012

[11] L Liu and T-G Kang ldquoMicroscopical identification and hier-archical cluster analysis of seven kinds of pilose antler velvetrdquoZhong Yao Cai vol 32 no 3 pp 345ndash347 2009

[12] D Chen Y Guo and W Ren ldquoCharacter identification of 12kinds of pilose antler medicinal materialsrdquo Zhong Yao Cai vol22 no 9 pp 441ndash444 1999

[13] G Xu C Liao X Ren et al ldquoRapid assessment of qualityof deer antler slices by using an electronic nose coupled withchemometric analysisrdquoBrazilian Journal of Pharmacognosy vol24 no 6 pp 716ndash721 2014

[14] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[15] G Xu X Wang C Liu et al ldquoAuthentication of official Da-huang by sequencing and multiplex allele-specific PCR of ashortmaturaseK generdquoGenome vol 56 no 2 pp 109ndash113 2013

[16] XWangG Xu C Liu et al ldquoDevelopment of deft amplificationrefractory mutation sequencing system (ARMSS) for discrimi-nating Pilos antler based on a short cytochrome b (Cytb) generdquoMitochondrial DNA 2014

[17] K Hsieh B S Ferguson M Eisenstein K W Plaxco and HT Soh ldquoIntegrated electrochemical microsystems for geneticdetection of pathogens at the point of carerdquo Accounts ofChemical Research vol 48 no 4 pp 911ndash920 2015

[18] D J Ye ldquoMicroscopic identification of pilose antler and deer-hornrdquo Zhong Yao Tong Bao vol 11 no 12 pp 17ndash19 1986

[19] Z Yan R Y Yuan C Y Wang Z H Zhang L P Cai and J KWang ldquoStudy on fingerprint of Corn cervi pantotrichum byHPCErdquo Food Science and Technology vol 34 no 4 pp 254ndash2582009

International Journal of

AerospaceEngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

RoboticsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Active and Passive Electronic Components

Control Scienceand Engineering

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

RotatingMachinery

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

Journal ofEngineeringVolume 2014

Submit your manuscripts athttpwwwhindawicom

VLSI Design

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Shock and Vibration

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Civil EngineeringAdvances in

Acoustics and VibrationAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Electrical and Computer Engineering

Journal of

Advances inOptoElectronics

Hindawi Publishing Corporation httpwwwhindawicom

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

SensorsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Modelling amp Simulation in EngineeringHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chemical EngineeringInternational Journal of Antennas and

Propagation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Navigation and Observation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

DistributedSensor Networks

International Journal of

6 Journal of Sensors

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nippon

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandus

Rangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandusRangifer tarandus

54

1519

65

24

33

10

100

8965

63

36

68

322952

5980

86

47

6951

89

66

001(a)

Cervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus nipponCervus nipponCervus nipponCervus nipponCervus nipponCervus nippon

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphus

Cervus elaphusCervus elaphusCervus elaphusCervus elaphus

Rangifer tarandus

89

231298

96

73

39297

91

74

77

64

81

3121

71

37

2150

54

100

001(b)

Figure 5 Cluster analysis ofDNAsequences (a)DNAsequences frommaterials of present study (b)DNAsequences fromGenbankdatabase

method based on high performance capillary electrophoresis(HPCE) [19] then had improved the objectivity and accuracynevertheless it is always not satisfied in terms of practicalityand identification power In this study electronic nose andDNA identification platform integrating electronic nose andDNA sequencing technology provides a more flexible andpractical identification platform Electronic nose is designedfor rapid screening while accurate identification basedon DNA method is performed when electronic nose failsto accurately identify some complex samples which doesincrease the efficiency of identification and vivify the use(Figure 1)

In fact many traditional Chinese medicines are aromaticsuch as herbal medicines from Umbelliferae and Labiataeand electronic nose is exactly suitable for rapid identification

of these materials Meanwhile DNA identification methodwhich is recognized as an accurate identification methoduniversally can make up for defects of electronic nose in caseof unclear identification

Electronic nose and DNA identification platform devel-oped in this study will also be improved according to thevision mentioned above in our further study We believe itwill play an important role in the field of TCM

4 Conclusions

In this study rapid identification of different velvet antlersbased on electronic nose showed the good identificationpower while accurate identification based on DNA sequenc-ing technology showed a correct identification rate of 100

Journal of Sensors 7

The results further proved that electronic nose and DNAidentification platform covering the advantages of electronicnose andDNA sequencing technology was effective for rapidand accurate identification and highly improved efficiencyand decreased cost of identification It is also believed thatelectronic nose and DNA identification platform will beof great significance to standardization normalization andmedication safety of Chinese medicine

Abbreviations

TCM Traditional Chinese medicineHPCE High performance capillary electrophoresisLDA Linear discriminant analysisCOI Cytochrome oxidase subunit I

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Xueyong Wang and Chunsheng Liu conceived and designedthe study Guojie Xu and Caili Liao accomplished the wholeexperiment and wrote the paper Xinyue Zhang Xiaorui Fuand Xiaolei Ren accounted for data analysis collecting sam-ples and processing samples All authors read and approvedthe final version of the paper

Acknowledgments

The authors thank Huihui Duan of Beijing University ofChinese Medicine for supporting the study This work wassupported by Beijing Natural Science Foundation of China(7152094 and 7142101) and National Natural Science Founda-tion of China (81274011)

References

[1] K-H Kim K-S Kim B-J Choi et al ldquoAnti-bone resorptionactivity of deer antler aqua-acupunture the pilose antler ofCervus koreanTEMMINCK var mantchuricus Swinhoe (Noky-ong) in adjuvant-induced arthritic ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 497ndash506 2005

[2] K-S Kim Y-H Choi K-H Kim et al ldquoProtective andanti-arthritic effects of deer antler aqua-acupuncture (DAA)inhibiting dihydroorotate dehydrogenase on phosphate ions-mediated chondrocyte apoptosis and rat collagen-inducedarthritisrdquo International Immunopharmacology vol 4 no 7 pp963ndash973 2004

[3] J-H Lin L-X Deng Z-Y Wu L Chen and L Zhang ldquoPiloseantler polypeptides promote chondrocyte proliferation via thetyrosine kinase signaling pathwayrdquo Journal of OccupationalMedicine and Toxicology vol 6 no 1 article 27 2011

[4] D Yan J Y Luo YM Han et al ldquoForensic DNA barcoding andbio-response studies of animal horn products used in tradi-tional medicinerdquo PLoS ONE vol 8 no 2 Article ID e558542013

[5] X Wang C Liu R Zhang L Huang and G Cui ldquoEstablish-ment of allele-specific diagnostic PCRmethod for identificationof zantlersrdquoZhongguo Zhongyao Zazhi vol 34 no 23 pp 3013ndash3016 2009

[6] R Zhang C-S Liu L-Q Huang X-Y Wang G-H Cui andL Dong ldquoStudy on the identification of Cornu Cervi Pan-totrichum with DNA barcodingrdquo Chinese Pharmaceutical Jour-nal vol 46 no 4 pp 263ndash266 2011

[7] M Natonek-Wioeniewska E Słota and B Kalisz ldquoUse ofcytochrome b polymorphism for species identification of bio-logical material derived from cattle sheep goats roe deer andred deerrdquo Folia Biologica vol 58 no 1-2 pp 47ndash50 2010

[8] Q Wang X Zhang H-Y Zhang et al ldquoIdentification of 12animal species meat by T-RFLP on the 12S rRNA generdquo MeatScience vol 85 no 2 pp 265ndash269 2010

[9] G D Haynes and E K Latch ldquoIdentification of novel singlenucleotide polymorphisms (SNPs) in deer (Odocoileus spp)using the BovineSNP50 Beadchiprdquo PLoS ONE vol 7 no 5Article ID e36536 2012

[10] Y H Kim E S Kim B S Ko et al ldquoA PCR-based assay fordiscriminating Cervus and Rangifer (Cervidae) antlers withmitochondrial DNA polymorphismsrdquo Journal of Animal Sci-ence vol 90 no 7 pp 2075ndash2083 2012

[11] L Liu and T-G Kang ldquoMicroscopical identification and hier-archical cluster analysis of seven kinds of pilose antler velvetrdquoZhong Yao Cai vol 32 no 3 pp 345ndash347 2009

[12] D Chen Y Guo and W Ren ldquoCharacter identification of 12kinds of pilose antler medicinal materialsrdquo Zhong Yao Cai vol22 no 9 pp 441ndash444 1999

[13] G Xu C Liao X Ren et al ldquoRapid assessment of qualityof deer antler slices by using an electronic nose coupled withchemometric analysisrdquoBrazilian Journal of Pharmacognosy vol24 no 6 pp 716ndash721 2014

[14] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[15] G Xu X Wang C Liu et al ldquoAuthentication of official Da-huang by sequencing and multiplex allele-specific PCR of ashortmaturaseK generdquoGenome vol 56 no 2 pp 109ndash113 2013

[16] XWangG Xu C Liu et al ldquoDevelopment of deft amplificationrefractory mutation sequencing system (ARMSS) for discrimi-nating Pilos antler based on a short cytochrome b (Cytb) generdquoMitochondrial DNA 2014

[17] K Hsieh B S Ferguson M Eisenstein K W Plaxco and HT Soh ldquoIntegrated electrochemical microsystems for geneticdetection of pathogens at the point of carerdquo Accounts ofChemical Research vol 48 no 4 pp 911ndash920 2015

[18] D J Ye ldquoMicroscopic identification of pilose antler and deer-hornrdquo Zhong Yao Tong Bao vol 11 no 12 pp 17ndash19 1986

[19] Z Yan R Y Yuan C Y Wang Z H Zhang L P Cai and J KWang ldquoStudy on fingerprint of Corn cervi pantotrichum byHPCErdquo Food Science and Technology vol 34 no 4 pp 254ndash2582009

International Journal of

AerospaceEngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

RoboticsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Active and Passive Electronic Components

Control Scienceand Engineering

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

RotatingMachinery

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

Journal ofEngineeringVolume 2014

Submit your manuscripts athttpwwwhindawicom

VLSI Design

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Shock and Vibration

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Civil EngineeringAdvances in

Acoustics and VibrationAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Electrical and Computer Engineering

Journal of

Advances inOptoElectronics

Hindawi Publishing Corporation httpwwwhindawicom

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

SensorsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Modelling amp Simulation in EngineeringHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chemical EngineeringInternational Journal of Antennas and

Propagation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Navigation and Observation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

DistributedSensor Networks

International Journal of

Journal of Sensors 7

The results further proved that electronic nose and DNAidentification platform covering the advantages of electronicnose andDNA sequencing technology was effective for rapidand accurate identification and highly improved efficiencyand decreased cost of identification It is also believed thatelectronic nose and DNA identification platform will beof great significance to standardization normalization andmedication safety of Chinese medicine

Abbreviations

TCM Traditional Chinese medicineHPCE High performance capillary electrophoresisLDA Linear discriminant analysisCOI Cytochrome oxidase subunit I

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Xueyong Wang and Chunsheng Liu conceived and designedthe study Guojie Xu and Caili Liao accomplished the wholeexperiment and wrote the paper Xinyue Zhang Xiaorui Fuand Xiaolei Ren accounted for data analysis collecting sam-ples and processing samples All authors read and approvedthe final version of the paper

Acknowledgments

The authors thank Huihui Duan of Beijing University ofChinese Medicine for supporting the study This work wassupported by Beijing Natural Science Foundation of China(7152094 and 7142101) and National Natural Science Founda-tion of China (81274011)

References

[1] K-H Kim K-S Kim B-J Choi et al ldquoAnti-bone resorptionactivity of deer antler aqua-acupunture the pilose antler ofCervus koreanTEMMINCK var mantchuricus Swinhoe (Noky-ong) in adjuvant-induced arthritic ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 497ndash506 2005

[2] K-S Kim Y-H Choi K-H Kim et al ldquoProtective andanti-arthritic effects of deer antler aqua-acupuncture (DAA)inhibiting dihydroorotate dehydrogenase on phosphate ions-mediated chondrocyte apoptosis and rat collagen-inducedarthritisrdquo International Immunopharmacology vol 4 no 7 pp963ndash973 2004

[3] J-H Lin L-X Deng Z-Y Wu L Chen and L Zhang ldquoPiloseantler polypeptides promote chondrocyte proliferation via thetyrosine kinase signaling pathwayrdquo Journal of OccupationalMedicine and Toxicology vol 6 no 1 article 27 2011

[4] D Yan J Y Luo YM Han et al ldquoForensic DNA barcoding andbio-response studies of animal horn products used in tradi-tional medicinerdquo PLoS ONE vol 8 no 2 Article ID e558542013

[5] X Wang C Liu R Zhang L Huang and G Cui ldquoEstablish-ment of allele-specific diagnostic PCRmethod for identificationof zantlersrdquoZhongguo Zhongyao Zazhi vol 34 no 23 pp 3013ndash3016 2009

[6] R Zhang C-S Liu L-Q Huang X-Y Wang G-H Cui andL Dong ldquoStudy on the identification of Cornu Cervi Pan-totrichum with DNA barcodingrdquo Chinese Pharmaceutical Jour-nal vol 46 no 4 pp 263ndash266 2011

[7] M Natonek-Wioeniewska E Słota and B Kalisz ldquoUse ofcytochrome b polymorphism for species identification of bio-logical material derived from cattle sheep goats roe deer andred deerrdquo Folia Biologica vol 58 no 1-2 pp 47ndash50 2010

[8] Q Wang X Zhang H-Y Zhang et al ldquoIdentification of 12animal species meat by T-RFLP on the 12S rRNA generdquo MeatScience vol 85 no 2 pp 265ndash269 2010

[9] G D Haynes and E K Latch ldquoIdentification of novel singlenucleotide polymorphisms (SNPs) in deer (Odocoileus spp)using the BovineSNP50 Beadchiprdquo PLoS ONE vol 7 no 5Article ID e36536 2012

[10] Y H Kim E S Kim B S Ko et al ldquoA PCR-based assay fordiscriminating Cervus and Rangifer (Cervidae) antlers withmitochondrial DNA polymorphismsrdquo Journal of Animal Sci-ence vol 90 no 7 pp 2075ndash2083 2012

[11] L Liu and T-G Kang ldquoMicroscopical identification and hier-archical cluster analysis of seven kinds of pilose antler velvetrdquoZhong Yao Cai vol 32 no 3 pp 345ndash347 2009

[12] D Chen Y Guo and W Ren ldquoCharacter identification of 12kinds of pilose antler medicinal materialsrdquo Zhong Yao Cai vol22 no 9 pp 441ndash444 1999

[13] G Xu C Liao X Ren et al ldquoRapid assessment of qualityof deer antler slices by using an electronic nose coupled withchemometric analysisrdquoBrazilian Journal of Pharmacognosy vol24 no 6 pp 716ndash721 2014

[14] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[15] G Xu X Wang C Liu et al ldquoAuthentication of official Da-huang by sequencing and multiplex allele-specific PCR of ashortmaturaseK generdquoGenome vol 56 no 2 pp 109ndash113 2013

[16] XWangG Xu C Liu et al ldquoDevelopment of deft amplificationrefractory mutation sequencing system (ARMSS) for discrimi-nating Pilos antler based on a short cytochrome b (Cytb) generdquoMitochondrial DNA 2014

[17] K Hsieh B S Ferguson M Eisenstein K W Plaxco and HT Soh ldquoIntegrated electrochemical microsystems for geneticdetection of pathogens at the point of carerdquo Accounts ofChemical Research vol 48 no 4 pp 911ndash920 2015

[18] D J Ye ldquoMicroscopic identification of pilose antler and deer-hornrdquo Zhong Yao Tong Bao vol 11 no 12 pp 17ndash19 1986

[19] Z Yan R Y Yuan C Y Wang Z H Zhang L P Cai and J KWang ldquoStudy on fingerprint of Corn cervi pantotrichum byHPCErdquo Food Science and Technology vol 34 no 4 pp 254ndash2582009

International Journal of

AerospaceEngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

RoboticsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Active and Passive Electronic Components

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Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

RotatingMachinery

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

Journal ofEngineeringVolume 2014

Submit your manuscripts athttpwwwhindawicom

VLSI Design

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Shock and Vibration

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Civil EngineeringAdvances in

Acoustics and VibrationAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Electrical and Computer Engineering

Journal of

Advances inOptoElectronics

Hindawi Publishing Corporation httpwwwhindawicom

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

SensorsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Modelling amp Simulation in EngineeringHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chemical EngineeringInternational Journal of Antennas and

Propagation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Navigation and Observation

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

DistributedSensor Networks

International Journal of

International Journal of

AerospaceEngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

RoboticsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Active and Passive Electronic Components

Control Scienceand Engineering

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

RotatingMachinery

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

Journal ofEngineeringVolume 2014

Submit your manuscripts athttpwwwhindawicom

VLSI Design

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Shock and Vibration

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Civil EngineeringAdvances in

Acoustics and VibrationAdvances in

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Electrical and Computer Engineering

Journal of

Advances inOptoElectronics

Hindawi Publishing Corporation httpwwwhindawicom

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

SensorsJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Modelling amp Simulation in EngineeringHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chemical EngineeringInternational Journal of Antennas and

Propagation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Navigation and Observation

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

DistributedSensor Networks

International Journal of