Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk

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  • Biosensors and Bioelectronics 26 (2011) 4753 4759

    Contents lists available at ScienceDirect

    Biosensors and Bioelectronics

    j our na l ho me page: www.elsev ier .co

    Develo enproges

    Laura Tra ereDepartment of avera

    a r t i c l

    Article history:Received 24 MReceived in reAccepted 25 MAvailable onlin

    Keywords:ProgesteroneFluorescent imQuantum dotsProgesteroneBioconjugates

    ts (Qnticajugaed tos werical r

    for pmmuMilk

    immlyte extraction. The minimum detectable level (IC10) of the developed immunoassay turned out to be0.1 ng/mL of progesterone in bovine milk. The sensitivity (IC50) achieved was 2.2 ng/mL with a repro-ducibility of 3.5% RSD as obtained from the results of the analysis of the triplicate of same samples butin three different days. Applicability of the proposed methodology was evaluated by analyzing cowsmilk samples enriched with known concentrations of progesterone and recoveries better than 90% were

    1. Introdu

    Accuratearticial inearly detecing reprodutime delay poor abilitying intervalDue to thosand accuraductive staet al., 2002)perature, rapedometricgoes withouthe purposein plasma o

    Progestethe female

    CorresponE-mail add

    0956-5663/$ doi:10.1016/j.achieved. 2011 Elsevier B.V. All rights reserved.


    oestrus detection is crucial for timed and successfulsemination and early detection of the pregnancy. Antion of a failed insemination is critical for maximis-ctive efciency, as it could allow a meaningful elapsedbefore a new repeated insemination. Furthermore, the

    to detect female animals in heat results in longer calv-s and lower milk production (Pennington et al., 1976).e reasons, reliable analytical methods to detect timelytely the occurrence of oestrus cycle and other repro-tes are needed (Friggens and Chagunda, 2005; Gillis. Several methods, including measurement of milk tem-diotelemetric measurement of vaginal temperature or

    technologies, have been used for oestrus prediction. Itt saying that the most effective and reliable method for

    is the direct determination of the level of progesteroner milk (Simersky et al., 2007).rone, a steroid hormone that is secreted in milk bymammals, is responsible of several activities related

    ding author. Fax: +34 985103474.ress: (A. Sanz-Medel).

    to reproductive cycle such as breast glandular development orthe preparation of the uterus for the pregnancy. Its concentra-tion in milk has a characteristic variation along the oestrus cycleand so progesterone is accepted as an ideal biomarker to con-trol the reproductive cows state, to detect the animal heat and todiagnose cows pregnancy (Kppel et al., 2007; Posthuma-Trumpieet al., 2009). It is well known that the onset of the heat is indi-cated by a rapid fall in the concentration of progesterone in milkto below 2-5 ng/mL. Furthermore, once the cow is pregnant theprogesterone concentration remains high and constant (Carraleroet al., 2007).

    Different methods have been developed for accurate determi-nation of progesterone in milk, including strategies based on thinlayer, gas or liquid chromatography coupled to mass spectrometrydetection (Darling et al., 1974; Daz-Cruz et al., 2003). However,such techniques are limited by several drawbacks including sub-stantial equipment costs and/or extensive and time-consumingsample pre-treatments, rendering progesterone routine deter-mination an expensive analysis. Alternatively, immunochemicalassays are the most popular approach nowadays for the determi-nation of progesterone (Gillis et al., 2006). A wide variety of labelshave been used in the development of progesterone immunoassaysincluding radioisotopes, enzymes or uorescent dyes (Byszewskaand Markiewicz, 2006; Colazo et al., 2008; Kppel et al., 2007) pro-

    see front matter 2011 Elsevier B.V. All rights reserved.bios.2011.05.044pment of a quantum dot-based uorescterone determination in bovine milk

    piella-Alfonso, Jose M. Costa-Fernndez, Rosario PPhysical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julian Cl

    e i n f o

    arch 2011vised form 16 May 2011ay 2011e 1 June 2011


    BSA conjugate

    a b s t r a c t

    The use of semiconductor quantum dosay for sensitive detection and quawater-soluble CdSe/ZnS QDs are conand a simple methodology is optimisgate. The obtained QD-linked antigenmonoclonal antibodies, as the biologQDs-based uorescent immunoassay

    After optimization, the developed ifrom 0.3 to 14.5 ng/mL in cow milk. directly analysed with the proposedm/locate /b ios

    t immunoassay for

    iro, Alfredo Sanz-Medel

    , 8, 33006 Oviedo, Spain

    Ds) as uorescent labels to develop a competitive immunoas-tion of progesterone in cows milk is described. Colloidalted to an antigen derivative (progesterone-BSA conjugate)

    determine the antigen concentration in the nal bioconju-e then employed together with unlabelled anti-progesteroneecognition elements, in the development of the quantitativerogesterone in bovine milk.noassay proved to cover a progesterone concentration rangesamples were just diluted 10-fold with deionised water andunoassay, without additional sample pre-treatment or ana-

  • 4754 L. Trapiella-Alfonso et al. / Biosensors and Bioelectronics 26 (2011) 4753 4759

    ducing optical or electrical signals that can be correlated with theconcentration of the analyte.

    In this vein, inorganic semiconductor uorescent nanocrystal-lites, also known as quantum dots (QDs), have emerged in the lastdecade as met al., 1998highly valuexcellent ain luminescto keep thethe use of tdevelop inndevelopmeQDs have bformats, suting (Bakaloimmunoass

    In this aQDs-based and quantiization of thcarried out of the antiging the chem(progesterobinding witto a compeple and conincubated iantibody. SestablishedQD label taimmunoass

    2. Materia

    2.1. Reagen

    All exper(see belowDeionised throughout

    2.1.1. Reagenanoparticle

    The CdSsized in outhe nanopamium oxidsolution in phine oxide(Milwaukeefrom Alfa Aegrade and csized QDs, wrespectively

    A seleniuby dissolvinproduce a ssulphide (Zsphere by mof diethyl z

    Water-san amphiphArgelles e

    maleic anhydride), dodecyl amine and tetrahydrofuran anhydrouswere purchased from Fluka (Basel, Switzerland) and Sigma Aldrich,respectively. The cross linker bis(6-aminohexyl)amine (Fluka)employed for the stabilization of the polymeric layer was stored


    Immu anAldr

    (Proed frlonabD

    (EDCfreezepared imine serck

    A 0.0was 0 mMthe iom tent.icon d, Spynthirecate b


    ite md, Stomephoreme

    (VaSIL qny)

    meaion cing, toac

    usi).eatina, S

    e nan insias setion,



    and asr 15PO.

    , 6 manocaterials with great potential in bioanalysis (Bruchez; Coto-Garca et al., 2011). Due to their innovative andable optoelectronic properties, QDs are becoming anlternative to the more conventional uorophore dyesent methodologies (Mattoussi et al., 2004). The ability

    functionality of biomolecules attached to QDs allowedhese nanomaterials as powerful luminescent labels toovative uorescent immunoassays. Since the pioneer

    nts in 1998 (Bruchez et al., 1998; Chan and Nie, 1998),een used to develop useful immunoassays in differentch as microarrays (Geho et al., 2005) or Western blot-va et al., 2005). However, very few direct quantitativeays using QDs have been developed so far.rticle the development and analytical evaluation of auorescent immunoassay for progesterone, detectioncation in milk is discussed. The synthesis and character-e water soluble colloidal nanoparticles of CdSe/ZnS wasrst. Then the QDs were bioconjugated to a derivativeen, the progesterone-BSA (Pro-BSA) conjugate, follow-istry of the carbodiimide. Considering that the antigen

    ne) is a hapten (small molecule that only has one site ofh the antibody), the immunoassay design was restrictedtitive format. Thus, free antigen present in the sam-stant (and known) amounts of labelled antigen weren a microtiter plate coated with a limited amount ofo, the competition for the antibody binding sites is. After that, the uorescence signal measurement of thekes place and the inhibition curve characteristic of theay is plotted.

    ls and methods

    ts and materials

    iments were carried out using analytical grade reagents) used as received without any further purication.ultrapure water (resistivity 18.2 M/cm) was used

    the work.

    nts used for the synthesis of the uorescentse/ZnS QDs, used as uorescent labels, were synthe-r laboratory. The precursors used for the synthesis ofrticles were selenium powder (100 mesh, 99.99%), cad-e (99.99%), hexamethyldisilathiane, 1.0 M diethyl zinchexane, trioctylphosphine (TOP, 90%) and trioctylphos-

    (TOPO, 99%), all of them purchased from Sigma Aldrich, WIS, USA) and hexylphosphonic acid (HPA) obtainedsar (Karlsruhe, Germany). The methanol HPLC gradient

    hloroform anhydrous (99%), used to purify the synthe-ere from Prolabo (Leuven, Belgium) and Sigma Aldrich,.m stock solution was prepared in an Ar-lled dry-boxg 1.63 mmol of selenium powder in 7.5 mL of TOP, toolution of trioctylphosphine selenide (SeTOP). A zincn/S/TOP) stock solution was also prepared in Ar atmo-ixing 1.18 mmol of hexamethyldisilathiane, 8.34 mmol

    inc and 10.25 mL of TOP.olubilisation of QDs was achieved by coating them withilic polymer synthesized in our laboratory (Fernndez-t al., 2007). For such purpose, poly(isobutylene-alt-

    at 4 C

    2.1.2. The

    Sigmajugateobtainmonocfrom Aimide in the was prand us

    Bovfrom Mtions. buffer

    A 5break QDs frsurem

    Am(Madriafter sthe indconjug

    2.2. In



    A hBarceloout thsphereow wmenta

    2.3. Pr

    2.3.1. CdS

    organo2001) of HPAneckedow foand TO270 Ction, nnological reagents and solutionstigen, 98% progesterone, was obtained fromich. The progesterone bovine serum albumin con--BSA), employed in the bioconjugation to the QDs, wasom AbD Serotec (Dusseldorf, Germany). The mousel anti-progesterone antibody (1 mg/mL) was purchasedSerotec. The ethyl-3-(dimethylaminopropyl) carbodi-) was purchased from Fluka and was stored at 18 Cer. For the QDs bioconjugation a solution 0.01 M of EDCed in 100 mM phosphate buffered saline (PBS) pH 7.4mediately.erum albumin (BSA), casein and powder milk obtained

    and Sigma Aldrich were compared as blocking solu-5% Tween 20 solution (SigmaAldrich) in PBS pH 7.4used as washing solution.

    borate buffer solution (SBB) at pH 12 was prepared tommunological interactions to allow dissolution of thehe immunoassay well plate before uorescence mea-

    Ultra-4 100 KDa centrifugal lters from Milliporeain) were used to purify the Pro-BSA-QD bioconjugatesesis. A Bradford reagent from Sigma was employed int estimation of progesterone concentration in the bio-ased on Bradford Test.


    icrotiter plates (96 wells) were obtained from Varianpain) and used in all immunoassays. The spec-tric measurements were carried out in a UV/Vis/NIRtometer (Perkin Elmer Lambda 900). The uorescentnts were performed in a spectrouorimeter Caryrian) equipped with a microplate reader module. Auartz cuvette (model 105.254-QS of Hellma, Mllheim,with 45 L volume was employed for spectrophoto-surements. To process the data obtained and t theurves a SOFTmax Pro software from Molecular DevicesGermany) was used.tivation of our synthesised CdSe/ZnS QDs was per-ng a 365 nm UV lamp (6 W, Vilbert Lourmat, Torcy,

    ng mantle with energy heater control (JP Selecta,pain) with simultaneous stirring was employed to carryoparticle synthesis. To keep a constant argon atmo-

    de the ask, a needle with argon ow was used. Thist to 25 mL/min by means of a rotameter (Serv Instru-

    Irigny, France).


    esis of CdSe/ZnS quantum dotsS QDs were synthesized using CdO as precursor via theallic route described by Pengs group (Peng and Peng,

    slight modications. Briey, 1.23 mmol of CdO, 0.6 g 17.5 g of TOPO were loaded into a 250 mL glass three-k. The mixture was heated up to 300320 C under argon20 min to allow the complete dissolution of CdO in HPAAfter cooling the temperature of the solution down toL of the SeTOP solution was swiftly injected. After injec-rystals were left to grow for about 11 min at 250 C. Once

  • L. Trapiella-Alfonso et al. / Biosensors and Bioelectronics 26 (2011) 4753 4759 4755

    the growth process has reached the desirable core size, 7.5 mL ofZn/S/TOP solution was added slowly at 230 C in order to allowthe generation of the shell, and then it was cooled down to 100 Cfor an hour. A schematic illustration is available in SupplementaryInformationlate the QDstep was imMeOH. The of 10 mL Meexcess of reFinally, thisby UV/VIS acles, their cwavelength

    The nadark.

    2.3.2. WateThe uo

    from organoorder to mapatible (wato achieve tbiomolecul(based on pprocedure The processand evaporawater-solub12.

    2.4. Milk sa

    A commprocessed) obtain the pmilk was sereal bovineterone.

    The applanalysis wafresh milk (be noted thtreatment twith Milli-Q

    3. Results

    Two difopment ofsensitive dmilk. One santi-progesuorescentcoated with(incubationtigated. Unprotocol anity achievedtoo high for

    Alternatconjugationof the antigthis approathe anti-proassay was b

    standards or samples containing progesterone and labelled antigen.Therefore, a bioconjugation of the antigen with the luminescentQDs, as labels, was needed.


    hapsrescee bioty oe.Ds a

    ntly thes toroced

    a bndezer shr fur


    of tt att

    tive oate) centeasee Prn is

    (Seeusedby vao ended um minten00 (Quriche ex. ConA conDa)altrl conhes)

    bioc to ev

    lum be s

    Prongthver iof thscopext, .


    optimncenfraction t Scheme S1. Once the synthesis was nished, to iso-s from the excess of the reaction media, a puricationplemented based on nanoparticles precipitation with

    solid, containing the QDs, was washed with three cyclesOH (and after each cycle it was centrifuged to removeagents) and then redispersed in anhydrous chloroform.

    colloidal solution was characterised spectroscopicallynd by uorescence. In this way the size of nanoparti-oncentration in the solution, the maximum emission

    and the size distribution could be assessed.l stock solution was stored at room temperature in the

    r solubilisation of QDsrescent QDs used in our experiments were synthesizedmetallic precursors and are inherently hydrophobic. Inke the nanoparticles hydrophilic to render them com-ter soluble) with bioanalytical applications, as well asheir bioconjugation in our laboratory with appropriatees, the QDs were coated with an amphiphilic polymeroly-maleic anhydride functional groups), following a

    recently described (Fernndez-Argelles et al., 2007). consists on a series of consecutive steps of additiontion of polymer and crosslinker solutions. The obtainedilised QDs were stored before use in 50 mMSBB at pH


    ercial bovine whole milk (ultra-high temperature, UHT,was employed for the immunoassay optimization and torogesterone calibration curves. Such commercial cowlected due to the high similitude...


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