Lactoferrin isolation using monolithic column coupled with spectrometric or micro-amperometric detector

Senso s 2008, 8, 464-487 senso s ISSN 1424-8220 © 2008 by MDPI www.mdpi.o g/senso s Full Pape Lac o e in Isola ion Using Monoli hic Column Coupled wi h Spec ome ic o Mic o-Ampe ome ic De ec o Voj ech Adam 1,2, Ond ej Zi ka 1, Pe Dolezal 2, Ladisla Zeman 2, Ales Ho na 3, Ja omi Hubalek 4, Jan Sileny 4, Sona K izko a 1, Libuse T nko a 5 and Rene Kizek 1,* 1 Depa men o Chemis y and Biochemis y, Mendel Uni e si y o Ag icul u e and Fo es y, Zemedelska 1, CZ-613 00 B no, Czech Republic 2 Depa men o Animal Nu i ion and Fo age P oduc ion, Facul y o Ag onomy, Mendel Uni e si y o Ag icul u e and Fo es y, Zemedelska 1, CZ-613 00 B no, Czech Republic 3 Depa men o Food Enginee ing, Facul y o Technology, Tomas Ba a Uni e si y, T.G. Masa yka 275, CZ-762 72 Zlin, Czech Republic 4 Depa men o Mic oelec onics, Facul y o Elec ical Enginee ing and Communica ion, B no Uni e si y o Technology, Udolni 53, CZ-602 00 B no, Czech Republic 5 Depa men Chemis y, Facul y o Science, Masa yk Uni e si y, Ko la ska 2, CZ-611 37 B no, Czech Republic * Au ho o whom co espondence should be add essed; e-mail:[email p o ec ed] Recei ed: 27 Decembe 2007 / Accep ed: 15 Janua y 2008 / Published: 24 Janua y 2008 Abs ac : Lac o e in is a mul i unc ional p o ein wi h an imic obial ac i i y and o he s o heal h bene icial p ope ies. The main aim o his wo k was o p opose easy o use echnique o lac o e in isola ion om cow colos um samples. P ima ily we u ilized sodium dodecyl sulpha e – polyac ylamide gel elec opho esis o isola ion o lac o e in om he eal samples. Mo eo e we es ed au oma ed mic o luidic Expe ion elec opho esis sys em o isola e lac o e in om he collos um sample. The well de eloped signal o lac o e in was de e mined wi h de ec ion limi (3 S/N) o 20 ng/ml. In spi e o he ac ha Expe ion is as e han SDS-PAGE bo h sepa a ion echniques canno be used in ou ine analysis. The e o e we ha e es ed hi d sepa a ion echnique, ion exchange ch oma og aphy, using monoli hic column coupled wi h UV-VIS de ec o (LC- UV-VIS). We op imized wa e leng h (280 nm), ionic s eng h o he elu ion solu ion (1.5 M NaCl) and low a e o he e en ion and elu ion solu ions (0.25 ml/min and 0.75 ml/min. espec i ely). Unde he op imal condi ions he de ec ion limi was es ima ed as 0.1 µg/ml o lac o e in measu ed. Using LC-UV-VIS we de e mined ha lac o e in concen a ion a ied om 0.5 g/l o 1.1 g/l in cow colos ums collec ed in he ce ain ime in e al up o Senso s 2008, 8 465 72 hou s a e bi h. Fu he we ocused on minia u iza ion o de ec ion de ice. We es ed ampe ome ic de ec ion a ca bon elec ode. The esul s encou aged us o a emp o minia u ise whole de ec ion sys em and o es i on analysis o eal samples o human aeces, because lac o e in le el in aeces is closely associa ed wi h he in lamma ions o in es ine mucous memb ane. Fo he pu pose o minia u iza ion we employed he echnology o p in ed elec odes. The de ec ion limi o lac o e in was es ima ed as 10 µg/ml measu ed by he sc een-p in ed elec odes ab ica ed by us. The ab ica ed elec odes we e compa ed wi h comme cially a ailable ones. I ollows om he ob ained esul s ha he esponses measu ed by comme cial elec odes a e app. en imes highe compa ed wi h hose measu ed by he elec odes ab ica ed by us. This phenomenon ela es wi h smalle wo king elec ode su ace a ea o he elec odes ab ica ed by us (abou 50 %) compa ed o he comme cial ones. The sc een-p in ed elec odes ab ica ed by us we e u ilized o de e mina ion o lac o e in aeces. Rega ding o ac ha sample o aeces was ob ained om young and heal hy man he amoun o lac o e in in sample was unde he limi o de ec ion o his me hod. Keywo ds: Lac o e in; Milk P o ein; Linea Sweep Vol amme y; Sc een-P in ed Ca bon Elec ode; Elec ochemis y; Liquid Ch oma og aphy; Monoli hic Column; UV-VIS Spec ome y 1. In oduc ion Milk as a sou ce o nu ien s con ains many biologically ac i e and in e es ing compounds. In 1939 a p o ein called lac o e in was disco e ed in cow milk. Mo e han wen y yea s la e his p o ein was i s ly isola ed om human b eas milk [1]. I s name is de i ed om i s o me classi ica ion as he mos impo an i on-binding p o ein in milk [2-5]. The molecula weigh and s uc u e o lac o e in is simila o o he i on-binding p o eins, ans e ins [6], he e o e i is some imes called lac o ans e in. Hololac o e in is consis ed om one linea polypep ide chain o ming wo sphe ical domains (C- and N- e minal), each domain con ains one i on binding si e. Lac o e in is a glycop o ein consis ed om less han se en hund ed aminoacid esidues e.g. 691 – human, 685 – pig. The p ima y s uc u e o lac o e in isola ed om milks o a ious animal species is e y simila . The highes con en s among aminoacids esidues ha e alanine (~10 %), leucine (~9 %) and glycine (~7 %), he lowes yp ophan (~1.5 %), his idine (~1.3 %) and me hionine (~0.6 %). The compa ison o aminoacid composi ion o lac o e in isola ed om eigh mammalian species is shown in Fig. 1 [7]. Senso s 2008, 8 466 Figu e 1. The compa ison o aminoacid composi ion o lac o e ine isola ed om human, bo ine, wa e bu alo, camel, goa , ho se, mouse and pig. The da a we e ob ained in da abase www.expasy.ch. 1.1 Biological unc ions o lac o e in Lac o e in is ound in mucosal sec e ions and g anules o neu ophils. I s highes le el was obse ed in colos um ollowed by milk, because du ing he lac a ion lac o e in concen a ion dec eases. Lac o e in can be also ound in exoc ine sec e ions such as ea s and nasal exuda e, sali a, b onchial mucus, gas oin es inal luids, ce ico aginal mucus and seminal luid [8-11]. The biological impo ance o lac o e in is s ill no ully clea [12-14]. I s an i logis ic, bac e ios a ic and bac e icidal e ec s a e assumed [15]. I s sec e ion in mucosals (mucous memb anes) can ha e ce ain e ec in p o ec i e incidence o BCG accine agains Mycobac e ium ube culosis [16,17]. In addi ion lac o e in modula es immune sys em and in es inal epide mal cells by posi i e e ec o g ow h o lac eous bac e ia (bi idobac e ia). Thanks o hese ac s lac o e in has exp essi e an imic obial e ec [18-22]. F om his oimmunological analyses o issues o pa ien s wi h acu e chole a highe le el o 024681012 Ala A g Asn Asp Cys Gln Glu Gly His Ile Leu Lys Me Phe P o Se Th T p Ty Val Sus sc o a Mus musculus Equus caballus Cap a hi cus Camelus d omeda ius Bubalus bubalis Bo in (Bos au us Human Amino acid composi ion (%) Senso s 2008, 8 467 his p o ein was ound in in es inal lamina p op ia [23]. Lac o e in was also used in he apy o Helicobac e pylo i in ec ion and his he apy led o pa hogen e adica ion [24,25]. An i i al e ec o lac o e in was obse ed in he e en o papiloma i uses ha a e cause s o ce ical cance [26] and a he pes simplex i us 2 [27]. Lac o e in was also used in he apy agains hepa i is C, whe e he educ ion o i al RNA i e was obse ed. Simila ly he usage o camel milk ( ha is empi ically used in adi ional medicines) led o he apeu ic e ec o hepa i is C i us [28,29]. I is in e es ing ha he lac o e in le el is s ic ly associa ed wi h he possibili y o emale HIV i us in ec ion [30-32]. Fo cu a i e pu poses he mos sui able and conside a e d ugs a e sea ched. As i is shown he pep ide as well as p o ein agmen s can be e y e ec i e oo. Expe imen ally he pep ide agmen s de i ed om lac o e in molecule we e used o supp ession o in ec ions caused by yeas s [33,34]. Mo eo e he in luence o milk p o eins on cance is s udied e y in ensi ely; i is possible o look o wa d o hei e ec o ca cinogenesis p ocesses. Based on he ecen ly published da a lac o e in inhibi s he umou s in small in es ine and in o he localiza ions. Lac o e in s a s he p ocesses leading o apop osis, inhibi s angiogenesis and modula es enzyme eac ions wi h ca cinogens [35,36]. I was disco e ed ha lac o e in in he combina ion wi h polyphenols can embody an ip oli e a i e e ec s. This ac was con i med on cell cul u e o ongue squamous ca cinoma (CAL-27). Combina ion o lac o e in wi h he polyphenols led o enhancing o p ocesses o p og ammed cell dea h o cance cells [37]. In s udy in 55 a m-houses in Pensyl ania (USA) i was ound ou ha he a e age lac o e in le el in analysed colos um was 0.8 mg/ml close o o al p o ein amoun 14.9 mg/ml. In addi ion o he subs ances, such as i amins, oils and mine als we e obse ed. Ob ained da a could assis in sea ching o possibili ies how o educe mo ali y and mo bidi y o na al cal es o how o moni o he milk quali y [38,39]. In he case o lac o e in addi ions in o pigs odde he p o ec i e e ec o hei immuni y was obse ed. The p o ec i e e ec was demons a ed as highe inc ease o weigh and lowe incidence o illnesses [40,41]. In addi ion he o al lac o e in le el in camel milk de e mined by ion exchange ch oma og aphy was 2.3 g/l [42]. Fu he analysis o compounds in sheep and goa milk we e published [43]. Because o he ac s desc ibed he einbe o e accu a e and p ecise isola ion o lac o e in om milk is needed. Own isola ion equi es pe manen milk in ake and i s con en e y a ies depending on many ac o s, mos o all on heal h s a e o he animals. Because o his ac he echniques based on molecula biology enabling con inual lac o e in p oduc ion by o he o ganisms a e e y ad an ageous. E en ecombinan human lac o e in p oduced by goa s wi h yield 2 g/l was p epa ed [44]. 1.2 Lac o e in and in es ine in lamma ion Endoscopy coupled wi h biopsy ep esen s he s anda d o in es ine in lamma ion de ec ion. This echnique is howe e e y expensi e and in asi e. Because o hese ac s he new echniques and ma ke s o in es ine in lamma ion de ec ion ha e been p oposing. Thei ad an age bases in he ac s ha hey enable us o p edic elapse o he in lamma ion and o moni o e ec o he ea men . Many o in lamma o y media o s such as leucocy es, cy okines and p o eins om neu ophil ac i a ion ha e been analysed in aeces. I was asce ained ha lac o e in le el has diagnos ic signi ica ion especially in he cases o in lamma ions a colon a ea (coli is). The lac o e in le el is signi ican ly inc eased in Senso s 2008, 8 468 he case o C ohn´s disease and may be excellen indica o o in lamma ion a colon a ea de e mined om aecal samples [45-51]. In addi ion he inc ease in lac o e in le el was obse ed a mo e han hal o pa ien s wi h colo ec al adenoca cinoma [49]. The enhanced lac o e in le el was de e mined immunohis ochemically a pa ien s wi h malignan umou s o kidneys compa ed wi h heal hy issues, whe e he le el o his p o ein was unde he de ec ion limi o he diagnos ic echnique used [52]. The unc ion o p o eomic esea ch is no only iden i ica ion o pa icula p o eins in he body o he o ganism bu also de e mina ion o hei unc ions ha a e e y equen ly connec ed wi h in e ac ions as DNA/p o ein, p o ein/p o ein, p o ein/RNA. The pape s epo ed on in es iga ion o in i o in e ac ion be ween lac o e in and coe uloplasmin we e ecen ly published [53,54]. 1.3 Lac o e in de ec ion Ve y ecen ly Enzyme-Linked ImmunoSo ben Assay (ELISA) and mass spec ome y was used o analysis o lac o e in [18]. Howe e hese e y accu a e echniques su e mos ly om high cos . The e o e he easy o use and low cos isola ion and de ec ion is sea ched. Lac o e in was isola ed by pa amagne ic pa icles wi h polyglycidyl-me haclya e connec ed wi h hepa ine. These pa icles showed high yield wi h maximum binding capaci y 164 mg o he a ge p o ein pe g am o he pa icles [55]. Ul a il a ion belongs o comme cially used echnique o lac o e in isola ion. This p ocess is double-s epped, in he i s s ep he ul a il a ion is ca ied ou and in second s ep as ion exchange ch oma og aphy is employed. The pu i y o he lac o e in is 94 % and eco e y 82 % [56]. In gene al ch oma og aphic echniques, mos o all he ion exchange ch oma og aphy, ep esen a way how o sepa a e lac o e in apidly a ela i ely low cos s [57-61]. F om he ion exchange ma e ials lac o e in can be isola ed by using o ca ionic su ac an (ce yldime hylammonium b omide) [62]. Howe e a e isola ion o lac o e in su icien ly sensi i e de ec ion echnique is needed. Elec ochemical de ec ion is an a ac i e al e na i e me hod o elec oac i e species de ec ion, because o i s inhe en ad an ages o simplici y, ease o minia u iza ion, high sensi i i y and ela i ely low cos [63-93]. The main aim o his wo k was o p opose easy o use echnique o lac o e in isola ion om cow colos um samples. Fu he we ocused on op imiza ion o minia u ized ampe ome ic de ice using sc een-p in ed elec odes o de e mina ion o he isola ed lac o e in. 2. Expe imen al Sec ion 2.1 Chemicals Ace oni ile and me hanol (HPLC-pu i y) we e ob ained om Me ck (Da ms ad , Ge many). The DMW lac o e in s anda d was pu chased om NUTRA ing ediens (Ne he lands). All o he chemicals used we e om Sigma-Ald ich (USA) unless o he wise indica ed. The s anda d s ock solu ions o lac o e in (concen a ion o 1 mg.ml-1) we e p epa ed in ACS wa e (Ald ich, USA) and s o ed in he da k a 4 °C. Wo king s anda d solu ions we e p epa ed daily by dilu ion o he s ock solu ions. The solu ions used o isola ion o lac o e in we e as ollows: wo king phospha e bu e (5 mM) pH 7.25; elu ion solu ion as 1.5 M NaCl p epa ed in he p esence o he wo king bu e ; egene a i e solu ion as 1 M NaOH. Senso s 2008, 8 469 2.2 Biological samples 2.2.1 Faeces The sample o aeces was ob ained om heal hy labo a o y s u . Sample (0.2 mg) was shaken in 1 ml o 0.1 M ace a e bu e , pH 5 (10 min, 300 pm, Genius, USA) and subsequen ly cen i uged. The supe na an ob ained was analysed. 2.2.2 Cow colos ums The samples o cow colos ums we e ob ained om a m in Žabčice (Czech Republic). Colos ums we e sampled a he ce ain ime in e al (1, 6, 12, 24, 36, 48, 60 and 72 h) a e bi h. The sample o colos um (abou 10 ml) was deep- ozen a –20°C p io o o he p ocessing. A e e eezing and s i ing 100 μl o high- iscose colos ums was pipe ed o 900 μl o he wo king phospha e bu e . Fu he he sample was s i ed o 30 s and cen i uged a 14 000 pm (He ich, Ge many) o 20 minu es. The supe na an was il e ed h ough 0.45 μm il e and en imes dilu ed by he wo king phospha e bu e . Then i was analyzed by liquid ch oma og aphy coupled wi h UV-VIS de ec o , chip capilla y elec opho esis – Expe ion and sodium dodecyl sulpha e – polyac ylamide gel elec opho esis. 2.3 Flow injec ion analysis wi h elec ochemical de ec ion A low injec ion analysis wi h elec ochemical de ec ion (FIA-ED) consis ed o sol en deli e y pump (Model 583 ESA Inc., Chelms o d, MA, USA), a gua d cell (Model 5020 ESA, USA), a eac ion coil (1 m) and an elec ochemical de ec o . The elec ochemical de ec o includes one low olume low- h ough analy ical cells (Model 5040, ESA, USA), which is consis ed o glassy ca bon wo king elec ode, hyd ogen-palladium elec ode as e e ence elec ode and auxilia y ca bon elec ode, and Coulochem III as a con ol module. The sample (5 μl) was injec ed manually. The ob ained da a we e p ocessed by CSW 32 so wa e. The expe imen s we e ca ied ou a oom empe a u e. Gua d cell po en ial was 0 V. A glassy ca bon elec ode was polished mechanically by 0.1 μm o alumina (ESA Inc., USA) and sonica ed a he oom empe a u e o 5 min using a Sono ex Digi al 10 P Sonica o (Bandelin, Be lin, Ge many) a 40 W [90,94]. 2.4 Linea sweep ol amme y a ca bon pas e elec odes Elec ochemical measu emen s we e pe o med wi h an Au olab analyze (EcoChemie, The Ne he lands) in connec ion wi h a VA-S and 663 (Me ohm, Swi ze land) using a s anda d cell wi h h ee elec odes. The elec ode sys em consis ed o a ca bon-pas e wo king elec ode, an Ag/AgCl/3 M KCl e e ence elec ode and glassy ca bon coun e elec ode. The ca bon pas e (abou 0.5 g) was made o g aphi e powde (Ald ich) and mine al oil (Sigma; ee o DNase, RNase, and p o ease). The a io o he g aphi e powde and mine al oil was 70/30; w/w. This pas e was housed in a Te lon body ha ing a 2.5-mm-diame e disk su ace. P io o measu emen s, he elec ode su ace was enewed by polishing wi h a so il e pape [66,70,95]. Ace a e bu e (0.1 M CH3COOH + 0.1 M CH3COONa, pH 5.0) was used as he suppo ing elec oly e. The analyzed samples we e deoxygena ed wi h a gon Senso s 2008, 8 470 (99.999%) sa u a ed wi h wa e o 120 s. All expe imen s we e ca ied ou a oom empe a u e. The linea sweep ol amme ic pa ame e s we e as ollows: ini ial po en ial o 0 V, end po en ial 1.5 V, s ep po en ial 5 mV. Smoo hing and baseline co ec ion was employed by GPES 4.4 so wa e supplied by EcoChemie. 2.5 Minia u ized ampe ome ic de ice using sc een p in ed elec odes The elec ochemical measu emen was ca ied ou wi h mul i-mode po en ios a BioS a (ESA, Inc. USA). I is ou -channel sys em wi h h ee ope a ing modes pe channel (amps, ol s, and emp). The sys em is connec ed h ough da a bus USB o pe sonal compu e . To he i s channel o he po en ios a he home made appa a us was connec ed. This appa a us consis s o basic pla e on which he connec o TX721 1115 wi h pins spacing 2.54 and he connec o 0039532035 om he manu ac u e Molex wi h pins spacing 1.25 mm a e placed. The connec o s a e designed o connec ion o wo di e en sc een-p in ed elec odes. Sc een-p in ed elec odes AC1.W5.R2 (BVT Technologies, Czech Republic) we e used. The sc een-p in ed elec ode includes wo king ca bon elec ode, sil e e e en elec ode wi h he laye o AgCl and coun e gold elec ode. Con ac s leading o he elec odes we e made om sil e , hei spacing was 2.54 mm. Dimensions o he senso we e 25.4 × 7.26 mm. Mo eo e we employed sc een-p in ed elec odes ab ica ed by us, o mo e de ails see in “Resul s and Discussion” sec ion. All elec ochemical measu emen s we e ca ied ou in B i on-Robinson bu e pH = 4.47 a oom empe a u e. Ampe ome ic measu emen was ca ied ou a po en ial 700 mV. 2.6 Liquid ch oma og aphy coupled wi h UV-VIS de ec o Flow sys em consis ed om pe is al ic pump Minipuls 3 (Gilson, F ance), ka ex CIM disc (BIA sepa a ions, Slo enia), UV de ec o (ESA, USA). One millili e o he biological sample ( aeces o cow colos um) was in oduced discon inuously o he sys em o one minu e using pe is al ic pump. Abso bance was measu ed a 280 nm. Fo o he expe imen al condi ions see “Resul s and Discussion” sec ion. 2.7 Chip capilla y elec opho esis – Expe ion Analyses on an au oma ed mic o luidic Expe ion elec opho esis sys em (Bio-Rad) we e ca ied ou acco ding o he manu ac u e ’s ins uc ions wi h supplied chemicals (Expe ion P o260 analysis ki , Bio-Rad). The sys em pe o ms elec opho esis (capilla y-based sepa a ion), s aining, des aining, band de ec ion (lase -induced luo escence) and imaging. The samples we e applied in o he Expe ion P o260 Chips. Expe ion So wa e was used o da a p ocessing. Each sample was dilu ed wi h wa e o he same p o ein concen a ion o 300 μg.mL-1, 4 μl aliquo s we e hen mixed wi h 2 μl o educing sample bu e , and a e 4 min o boiling, 84 μl o wa e was added. A e p iming o he chip wi h gel and gel-s aining solu ion in he dilu ed p iming s a ion sample, he mix u e (6 μl) was loaded in o sample wells. The P o260 Ladde included in he ki was used as a s anda d. Senso s 2008, 8 471 2.8 Sodium dodecyl sulpha e - polyac ylamide gel elec opho esis Sodium dodecyl sulpha e – polyac ylamide gel elec opho esis (SDS-PAGE) was pe o med acco ding o Laemmli [96] using a Biome a maxigel appa a us (Biome a, Ge many). Fi s 10% (w/ ) unning, hen 5% (w/ ) s acking gel was pou ed, he gels we e p epa ed om 30% (w/ ) ac ylamide s ock solu ion wi h 1% (w/ ) bisac ylamide concen a ion; he polyme iza ion o he unning gel was ca ied ou a oom empe a u e o 1 h and 30 min o he s acking gel. P io o analysis he samples we e mixed wi h sample bu e con aining 5% ( / ) 2-me cap oe hanol in a 1:1 a io. The samples we e boiled o 2 min. and hen loaded on o a gel in 20 μl aliquo s. Fo de e mina ion o molecula weigh , he p o ein ladde “P ecision plus p o ein s anda ds” om Bio ad was used. The elec opho esis was un a 120V wi h cooling o 5 hou s. Coomassie blue s aining o he gels was pe o med acco ding o Diezel e al. [97]. The gels we e also s ained by sil e . A e he s aining, he gel was scanned and analyzed using Bioligh so wa e (Vilbe -Lou ma , Ge many). 2.9 S a is ical analysis MICROSOFT EXCEL® (USA) was used o s a is ical analyses. Resul s a e exp essed as mean ± S.D. unless no ed o he wise. Di e ences wi h p < 0.05 we e conside ed signi ican . The de ec ion limi s (3 S/N) we e calcula ed acco ding o Long [98], whe eas N was exp essed as s anda d de ia ion o noise de e mined in he signal domain. The o he app oaches o o es ima ion o de ec ion limi s we e epo ed by La agnini e al. [99]. 3. Resul s and Discussion 3.1 SDS-PAGE and Expe ion analysis o lac o e in Lac o e in as p o ein wi h high molecula weigh is mino cons i uen o eal samples like cow milk. As i is men ioned in “In oduc ion” sec ion, lac o e in has many bene icial biological e ec s o men’s and animal’s heal h. To isola e he p o ein om a sample sepa a ion echnique wi h maximum eco e y and abili y o pu e he p oduc is needed. Milk is he mos impo an sou ce o lac o e in, bu i con ains conside able amoun s o o he p o eins as i is shown in Fig. 2. P ima ily we employed SDS-PAGE and Expe ion o isola e lac o e in om he collos um sample p epa ed as desc ibed in “Expe imen al sec ion”. A he end o he sepa a ion Coomassie blue s aining ollowed and he exp essi e band o lac o e in a molecula weigh 77 kDa was de ec able (Fig. 2A line 2 and 3). Simila ly o Coomassie blue s aining i we s ained by sil e , he majo i y bands o lac o e in we e also isible (Fig. 2B). Howe e SDS-PAGE canno be u ilized o ou ine analysis due o labou ing and ime consuming p epa a ion o he gel, lowe sensi i i y and ha d ob aining o he ac ion wi h lac o e in om he gel. Mo eo e we es ed Expe ion o isola e lac o e in om he collos um sample. The esul o he elec opho e ic analysis is shown in Fig. 2C. The well de eloped signal o lac o e in was de e mined. In spi e o he ac ha Expe ion is as e han SDS-PAGE he lack o using o Expe ion o ou ine analysis is low olume o a sample, he e o e low amoun s o isola ed laco o e in we can ge . Besides ha bo h echnique shown ha many o he p o eins a e p esen ed in he collos um samples (Fig. 2). Senso s 2008, 8 472 3.1 Lac o e in sepa a ion using monoli hic column Due o ou ine applica ion, we ha e es ed hi d sepa a ion echnique, ion exchange ch oma og aphy, using monoli hic column. The samples o colos ums we e p epa ed as desc ibed in “Expe imen al sec ion” and analysed by using liquid ch oma og aphy coupled wi h UV-VIS de ec o (LC-UV-VIS). One millili e o he cow colos um was in oduced discon inuously o he sepa a ion sys em consis ing o pe is al ic pump Minipuls 3 (Gilson, F ance), ca ion exchange CIM disc in monoli hic column (BIA sepa a ions, Slo enia) and UV/VIS de ec o (ESA, USA). The scheme o he ins umen is shown in inse in Fig. 3. The p epa ed colos um sample was sepa a ed using monoli hic column. P o eins sepa a ion on CIM disc con aining –SO3 g oups is based on he change o he p o eins cha ges a di e en pH and ionic s eng h. I is a common knowledge ha isoelec ic poin (pI) is he pH, a which a pa icula molecule ca ies no ne elec ical cha ge. I he alue o he pH is highe han pI o p o ein, his p o ein has gene al nega i e cha ge. This e ec is used in ca ion exchange ch oma og aphy on monoli hic column. Nex ad an age o he monoli hic disc is i s high po osi y and consequen ly ela i ely low wo king p essu e o which he column as well as compounds in he column a e exposed. Figu e 2. SDS-PAGE wi h (A) Coomassie blue s aining o (B) Sil e s aining and (C) Expe ion capilla y elec opho esis o ladde , lac o e in s anda d (100 µg/ml) and milk sample Fo he isola ion o lac o e in he ollowing p ocess was used. A e he in oducing o he sample he inle was econnec ed om eal sample o s ock solu ion o he wo king phospha e bu e ( e en ion solu ion) and he bu e was wen y minu es pumped h ough he sys em a low a e o 0.75 ml/min o Ladde Collos um Lac o e in 15 20 25 37 50 75 100 150 250 Molecula weigh (kDa) Ladde Collos um Lac o e in Coomassie blue s aining Sil e s aining Uppe ma ke Lac o e in Sys em peak Lowe ma ke 1.2 10 20 25 37 50 75 100 150 260 Capilla y elec opho esis Uppe ma ke Lac o e in Sys em peak Lowe ma ke 1.2 10 20 25 37 50 75 100 150 260 Capilla y elec opho esis ABC Ladde Lac o e in Collos um Molecula weigh (kDa) Senso s 2008, 8 479 10A). The highes change in he signal heigh was obse ed be ween 650 and 750 mV. Maximal cu en esponse o lac o e in was measu ed a 750 mV (Fig. 10A). This po en ial was used in he ollowing expe imen s, in which we a emp ed o compa e he ab ica ed sc een-p in ed elec odes wi h comme cially ones supplied by BVT Technologies. Using bo h ypes o he elec odes we measu ed calib a ion cu e (Figs. 10B,C). I clea ly ollows om he esul s ob ained ha lac o e in can be analysed using bo h elec odes. The dependence o signal heigh on lac o e in concen a ion (calib a ion cu e) measu ed by he comme cial elec odes is shown in Fig. 10B. The plo ed equa ion was y = 0.0871x + 0.6658; R2 = 0.9787 wi h ela i e s anda d de ia ion 6 %. The de ec ion limi (3 S/N) o lac o e in was es ima ed as 1 µg/ml. In he case o usage o he sc een-p in ed elec odes ab ica ed by us he calib a ion cu e had he equa ion y = 0.01x + 0.0778; R2 = 0.9730. The de ec ion limi (3 S/N) o lac o e in was es ima ed as 10 µg/ml (Fig. 10B). I ollows om he esul s ob ained ha he esponses measu ed by comme cial elec odes a e app. en imes highe compa ed wi h hose measu ed by he elec odes ab ica ed by us. This phenomenon ela es wi h smalle wo king elec ode su ace a ea o he elec odes ab ica ed by us (abou 50 %) compa ed o he comme cial ones. Figu e 10. Ampe ome ic lac o e in de ec ion a sc een-p in ed ca bon elec odes. (A) Hyd odynamic ol ammog am o lac o e in (100 µg/ml) measu ed using he sc een- p in ed elec odes ab ica ed by us. Dependence o lac o e in signal heigh on i s concen a ion measu ed (B) by he comme cial elec odes BVT and/o (C) by elec odes ab ica ed by us. In inse s o B and C: eal lac o e in signals measu ed by he elec odes. Wo king po en ial 750 mV (B,C), olume o he suppo ing elec oly e (0.1 M ace a e bu e , pH 5.0) 10 µl, sample olume 5 µl. 3.8 Lac o e in de ec ion in aeces samples The sc een-p in ed elec odes ab ica ed by us we e u ilized o de e mina ion o lac o e in aeces. Sample (0.2 mg) was shaken in 1 ml o 0.1 M ace a e bu e , pH 5 and subsequen ly cen i uged. The 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0255075100 0 1 2 3 4 5 6 7 8 9 10 0255075100 Lac o e in concen a ion (µg/ml) Peak heigh (nA) comme cial elec odes minia u ized elec odes 1 nA 10 s 0.2 nA 0 20 40 60 80 100 120 300 500 700 900 1100 Applied po en ial (V) Peak heigh (nA) Lac o e in concen a ion (µg/ml) Peak heigh (nA) 10 s ABC Senso s 2008, 8 480 lac o e in in he supe na an ob ained was isola ed using monoli hic column. The isola ed lac o e in was measu ed using he sc een-p in ed elec odes. Rega ding o ac ha sample o aeces was ob ained om young and heal hy man he amoun o lac o e in in sample was unde he limi o de ec ion o his me hod. Due o his ac he sample was spiked by he di e en quan i ies o lac o e in. P ima ily we u ilized LC-UV-VIS ins umen o de ec he spiked lac o e in (Fig. 11A). As i is shown in Fig. 11B, lac o e in was de ec able in he sample o he lac o e in spiked aeces also by he sc een-p in ed elec odes. The ob ained ampe ome ic esponses we e well de eloped and de ec able (Fig. 11B). Figu e 11. Isola ion o lac o e in using monoli hic column in sample o aeces spiked by 0, 12.5, 25, 50 and 100 µg/ml o he a ge p o ein. (A) Typical ch oma og ams o spiked lac o e in o aces sample measu ed a 280 nm by LC-UV-VIS. (B) Ampe ome ic lac o e in de ec ion a he sc een-p in ed elec odes ab ica ed by us. 4. Conclusion Resea ch and de elopmen in his o p oposing new senso s and biosenso s is wide and mul idisciplina y, spanning biochemis y, bio eac o science, physical chemis y, elec ochemis y, elec onics and so wa e enginee ing. Mos o his cu en endea ou conce ns po en iome ic and ampe ome ic biosenso s and colo ime ic pape enzyme s ips [77,82-85,88]. He e we epo on 0 2 4 6 8 10 12 14 16 012.52550100 Peak heigh (nA) Spiked lac o e in concen a ion (µg/ml) 10 s 1 nA Human aeces sample o aeces 10 AU (280 nm) 1 min Lac o e in 50 μg/ml 10 μg/ml 5 μg/ml sample o aeces 10 AU (280 nm) 1 min1 min Lac o e in 50 μg/ml 10 μg/ml 5 μg/ml AB Senso s 2008, 8 481 coupling o monoli hic column sepa a ion and elec ochemical de ec ion a he sc een-p in ed elec odes as a ool o de e mina ion o lac o e in. Acknowledgemen s We g a e ully acknowledge he G an Agency o he Czech Republic (g an No. GACR 102/08/1546) o he inancial suppo o his wo k. Re e ences 1. Bezko o ainy, A. Human milk and colos um p o eins - e iew. J. Dai y Sci. 1977, 60, 1023- 1037. 2. Sanchez, L.; A anda, P.; Pe ez, M.D.; Cal o, M. Concen a ion o Lac o e in and T ans e in Th oughou Lac a ion in Cows Colos um and Milk. Biol. Chem. Hoppe-Seyle 1988, 369, 1005- 1008. 3. Sanchez, L.; Cal o, M.; B ock, J.H. Biological Role o Lac o e in. A ch. Dis. Child. 1992, 67, 657-661. 4. 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