An Organic/Inorganic Hybrid Membrane as a Solid “Turn-On” Fluorescent Chemosensor for Coenzyme A (CoA), Cysteine (Cys), and Glutathione (GSH) in Aqueous Media

Senso s 2012, 12, 2969-2982; doi:10.3390/s120302969 senso s ISSN 1424-8220 www.mdpi.com/jou nal/senso s A icle An O ganic/Ino ganic Hyb id Memb ane as a Solid “Tu n-On” Fluo escen Chemosenso o Coenzyme A (CoA), Cys eine (Cys), and Glu a hione (GSH) in Aqueous Media Saúl Vallejos, Ped o Es é ez, Sa u nino Ibeas, Félix C. Ga cía, Felipe Se na and José M. Ga cía * Depa amen o de Química, Facul ad de Ciencias, Uni e sidad de Bu gos, Plaza de Misael Bañuelos s/n, E-09001 Bu gos, Spain; E-Mails: s [email protected] (S.V.); [email p o ec ed] (P.E.); [email p o ec ed] (S.I.); [email p o ec ed] (F.C.G.); [email p o ec ed] (F.S.) * Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed]; Tel.: +34-947-258-085; Fax: +34-947-258-831. Recei ed: 17 Janua y 2012; in e ised o m: 23 Feb ua y 2012 / Accep ed: 23 Feb ua y 2012 / Published: 2 Ma ch 2012 Abs ac : The p epa a ion o a luo ogenic senso y ma e ial o he de ec ion o biomolecules is desc ibed. S a egic unc ionalisa ion and copolyme isa ion o a wa e insoluble o ganic senso y molecule wi h hyd ophilic comonome s yielded a c osslinked, wa e -swellable, easy- o-manipula e solid sys em o wa e ‘‘dip-in’’ luo ogenic coenzyme A, cys eine, and glu a hione de ec ion by means o hos -gues in e ac ions. The senso y ma e ial was a memb ane wi h gel-like beha iou , which exhibi s a change in luo escence beha iou upon swelling wi h a wa e solu ion o he a ge molecules. The memb ane ollows a “ u n-on” pa e n, which pe mi s he i a ion o he abo emen ioned biomolecules. In his way, he wa e insoluble sensing mo i can be exploi ed in aqueous media. The senso y mo i wi hin he memb ane is a chemically ancho ed pipe azinedione- de i a i e wi h a weakly bound Hg(II). The esponse is caused by he displacemen o he ca ion om he memb ane due o a s onge complexa ion wi h he biomolecules, hus eleasing he luo escen senso y moie ies wi hin he memb ane. Keywo ds: senso y ma e ials; chemosenso ; luo ogenic senso ; biomolecules; sensing biomolecules OPEN ACCESS Senso s 2012, 12 2970 1. In oduc ion The de elopmen o sensing molecules o he de ec ion o chemicals is a opic o cu en in e es [1–6]. The ecogni ion o a ge molecules based on he a ia ion o a mac oscopic p ope y o a sensing molecule associa ed wi h he speci ic in e ac ions o he a ge wi h he ecep o mo i s o he senso can be used o p epa e senso y solu ions o he easy, cheap and apid quan i ica ion o chemicals by means o a widely used analy ical echnique (e.g., UV/Vis and/o spec o luo ome y). Mo eo e , i he ecep o and he ansducing mo i s a e chemically bound o a polyme ne wo k s uc u e, hen he o ganic ma e ial can be desc ibed as a solid sys em, which can po en ially be used as a solid ki o he ‘‘dip-in’’ de ec ion o analy es [3]. Fo medical, biomedical and en i onmen al easons, biological molecules a e among he mos impo an a ge analy es [7]. Biomolecules con aining a hiol g oup, such as coenzyme A (CoA), L-cys eine (Cys), and glu a hione (GSH), play impo an oles in biological p ocesses including acyl g oup ca ie capabili y o oxida ion/ educ ion acili y (i.e., in amolecula educ ion-oxida ion me abolic cycles), which occu in hund eds o biochemical eac ions [8–12]. The de e mina ion o he biomolecule concen a ion has been unde aken wi h a ious me hodologies. One o he mos in e es ing is he sensing me hodology based on he hos -gues sup amolecula app oach. The sup amolecula app oach has ecen ly been applied o he de e mina ion o Cys [13–27], CoA [8–10], and GSH o a lesse ex en [28]. He ein, we desc ibe a senso y o ganic/ino ganic hyb id memb ane o he luo ogenic de ec ion o h ee impo an biomolecules: CoA, Cys, and GSH ( he chemical s uc u es a e shown in Scheme 1). The memb ane was a dense ilm consis ing o a hyd ophilic ac ylic ne wo k ha con ained a small amoun o pipe azinedione-de i a i e/Hg(II) moie ies as he senso y mo i owa d he biomolecules men ioned abo e. The pipe azinedione-de i a i e was chemically ancho ed o he copolyme backbone. The wa e -swelled memb ane esponded o he p esence o he a ge s in an aqueous en i onmen a physiological pH wi h an inc ease in he luo escence in ensi y (i.e., a luo escence “ u n-on” pa e n), which pe mi ed he i a ion o he biomolecules. Scheme 1. S uc u e o Cys, GSH, and CoA. H2NOH O SH HOOC NNCOOH NH2 OHS HO H Cys eine (Cys) Glu a hione (GSH) O OHO POO - OH N NN N NH2 HS NN O O HH O OH CH3 H3C POPO OO O-O- Coenzyme A (CoA) Senso s 2012, 12 2971 2. Expe imen al Sec ion 2.1. Ma e ials The ollowing comme cially a ailable ma e ials and sol en s we e used as ecei ed, unless o he wise indica ed: me cu y(II) ace a e (Sigma Ald ich, 98%), glycine (Sigma Ald ich, 99%), me hac yloyl chlo ide (Fluka, 97%), e hylene glycol dime hac yla e (Ald ich, 98%), e hylene glycol (Fluka), ace ic anhyd ide (Sigma Ald ich, pu iss.), po assium -bu oxide (Sigma Ald ich, 99.99%), 4-(dime hylamino)benzaldehyde (Sigma Ald ich, 98%), ie hylamine (Fluka, 99.5%), li hium chlo ide (Sigma Ald ich, 99%), 4-ni obenzaldehyde (Sigma Ald ich, 99%), sodium sulphide nonahyd a e (Sigma Ald ich, 98%), dioxane (P obus, 99%), 1- inyl-2-py olidone (Sigma Ald ich, 99%), N-me hyl-2-py olidone (Sigma Ald ich, 99.5%), die hyl e he (VWR, 99.99%), DMSO (Me ck, 99%), ace one (Ald ich, 99%), e hanol (Ald ich, 99%), me hanol (VWR, o HPLC), DMF (Ald ich, 99%), coenzyme A ili hium sal (Calbiochem, 99.9%), L-glu a hione educed (Al a Aesa , 97%), and L-cys eine hyd ochlo ide monohyd a e (VWR). Azo-bis-isobu y oni ile (AIBN, Fluka, 98%) was ec ys allised wice om me hanol. 2.2. Measu emen s 1H and 13C-NMR spec a we e eco ded in deu e a ed dime hyl sulphoxide (DMSO-d6) as he sol en using a Va ian Ino a 400 spec ome e ope a ing a 399.92 and 100.57 MHz, espec i ely. In a ed spec a (FTIR) we e eco ded wi h a Nicole Impac spec ome e o wi h a JASCO FT/IT-4100 i ed wi h a PIKE TECH “Mi acle” ATR. The mog a ime ic analysis (TGA) da a we e eco ded using 5 mg o sample unde a ni ogen o oxygen a mosphe e on a TA Ins umen Q50 TGA analyze a a scan a e o 10 °C min−1. UV-Vis spec a we e eco ded using a Va ian Ca y3-Bio UV-Vis spec opho ome e . The luo escence spec a we e eco ded using a Va ian Ca y Eclipse luo ome e . Millipo e-Q wa e was used o p epa e he solu ions. To de e mine he ensile p ope ies o he memb anes, s ips (5 mm in wid h, 30 mm in leng h, and 30–45 μm hick) we e cu om he polyme ilms and measu ed using a Houns ield H10KM Uni e sal Tes ing Dynamome e a 20 °C. Mechanical clamps held he sample, and an ex ension a e o 5 mm min−1 was applied using a gauge leng h o 10 mm. A leas six samples we e es ed o each polyme , and he da a we e a e aged. 2.3. In e media es and Monome Syn hesis The o e all syn he ic s eps o he monome a e shown in Scheme 2. Syn hesis o 1,4-diace ylpipe azine-2,5-dione (1). Glycine (a o al o 100 g, 1.33 mol) was dissol ed in e hylene glycol (500 mL) in a 1,000 mL lask i ed wi h a mechanical s i e . The mix u e was s i ed a 170 °C o 3 h, and he solu ion was cooled a 5 °C o 20 h. The p ecipi a e, pipe azine-2,5- dione, was collec ed by il a ion and washed wi h me hanol (500 mL). Then, he solid was dissol ed in boiling wa e , and he solu ion was cooled o e nigh . The whi e p oduc was il e ed o and washed wi h me hanol. Yield: 30%. M.p.: 330 °C. 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 4.64 (2H, s, NH); 3.85 (4H, s, CH2). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 168.46, 43.83. EI-LRMS m/z: Senso s 2012, 12 2972 114 (M+●, 100), 86 (8), 72 (2), 58 (5), 56 (7). FTIR [wa enumbe s (cm−1)]: νN-H: b oadband (3,250, 2,750); νC=O: 1,696. Pipe azine-2,5-dione (18.7 g, 0.164 mmol) and ace ic anhyd ide (85 mL) we e added o a 250 mL lask equipped wi h a e lux condense . The mix u e was s i ed a e lux o 7 h. The sol en was emo ed by dis illa ion. The p oduc 1 was washed wi h die hyl e he and collec ed by il a ion. Yield: 90%. M.p.: 96 °C. 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 4.64 (6H, s, CH3); 3.84 (4H, s, CH2). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 173.61, 168.17, 43.32, 26.12. EI-LRMS m/z: 198 (M+●, 30), 156 (41), 114 (47), 86 (3), 71 (30), 43 (12). FTIR [wa enumbe s (cm−1)]: νN-C=O: b oadband (3,452, 3,365); νC=O: 1,718. Syn hesis o (3Z,6Z)-3-(4-(dime hylamino)benzylidene)-6-(4-ni obenzylidene) pipe azine-2,5-dione (2). A lask equipped wi h a e lux condense was cha ged wi h 1,4-diace ylpipe azine-2,5-dione (1, 4.94 g, 25 mmol) and 4-(dime hylamino)benzaldehyde (3.72 g, 25 mmol) which we e dissol ed in DMF (70 mL). Po assium -bu oxide (2.8 g, 25 mmol) was added, and he mix u e was s i ed a oom empe a u e o 12 h. The p oduc , (Z)-3-(4-(dime hylamino)benzylidene)-1-ace ylpipe azine-2,5- dione, was p ecipi a ed in wa e and collec ed by il a ion. Finally, he solid was washed wi h wa e and me hanol. Yield: 50%. M.p.: 210 °C. 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 10.19 (1H, s, NH); 7.53 (2H, d, J 8.7, A H); 6.95 (1H, s, CH); 6.79 (2H, d, J 9.0, A H); 4.39 (2H, s, CH2); 3.02 (6H, s, CH3); 2.51 (3H, s, CH3). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 172.31, 165.00, 163.19, 151.28, 132.07, 122.99, 122.45, 120.86, 112.39, 46.08, 26.96. EI-LRMS m/z: 287 (M+●, 90), 245 (100), 160 (52), 115 (3), 78 (11), 62 (14). FTIR [wa enumbe s (cm−1)]: νN-H: b oadband (3,661, 3,310); νC=O: 1,696, 1,597 and 1,521. (Z)-3-(4-(dime hylamino)benzylidene)-1-ace ylpipe azine-2,5-dione (2.6 g, 9 mmol) and 4-ni o- benzaldehyde (1.36 g, 9 mmol) we e dissol ed in DMF (135 mL) in a lask equipped wi h a e lux condense . T ie hylamine (0.9 g, 9 mmol) was added, and he mix u e was s i ed a 130 °C o 12 h. A solid was collec ed by il a ion and washed wi h me hanol. Finally, he solid was washed wi h ace one a i s e lux empe a u e in a lask equipped wi h a e lux condense . Yield: 50%. M.p.: no obse ed ( he compound was amo phous; howe e , an exo he mic c ys allisa ion peak was obse ed a 353 °C). 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 10.43 (1H, s, NH); 10.09 (1H, s, NH); 8.20 (2H, d, J 6.42, A H); 7.75 (2H, d, J 7.35, A H); 7.43 (2H, d, J 6.42, A H); 6.74 (4H, , J 5.93, A H); 2.95 (6H, s, CH3). EI-LRMS m/z: 378 (M+●, 100), 332 (3), 287 (1), 215 (1), 159 (37), 117 (5), 89 (4), 77 (1). FTIR [wa enumbe s (cm−1)]: νN-H: b oadband (3,628, 3,331), νN-H: 3,211; νC=O: 1,674 and 1,626; νNO: 1,578 (asymme ic) and 1,339 (symme ic). Syn hesis o N-(4-((1Z)-((Z)-5-(4-(dime hylamino)benzylidene)-3,6-dioxopipe azin-2-ylidene)me hyl) phenyl)me hac ylamide (3). In a 250 mL lask i ed wi h a e lux condense , compound 2 (1.8 g, 4.75 mmol) was dissol ed in dioxane (100 mL). Sodium sulphide nonahyd a e (3.43 g, 14.25 mmol) was added o he solu ion, and he mix u e was s i ed a 80 °C o 24 h. The solu ion was il e ed, and wa e (400 mL) was added. The esul an p ecipi a e, (3Z,6Z)-3-(4-(dime hylamino) benzylidene)-6-(4- aminobenzylidene) pipe azine-2,5-dione, was il e ed o and washed wice wi h me hanol. Yield: 72%. M.p.: 307 °C. 1 H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 9.81 (2H, s, NH2); 7.47 (2H, d, J 9.03, A H); 7.32 (2H, d, J 9.03, A H); 6.78 (2H, d, J 10.08, A H); 6.69-6.61 (4H, m, A H); 5.63 (2H, s, NH); 3.00 (6H, s, CH3). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 159.26, 159.12, 150.66, 150.08, 131.66, 131.43, 123.50, 122.82, 121.29, 120.99, 117.10, 116.41, 114.36, 112.60. EI-LRMS Senso s 2012, 12 2973 m/z: 348 (M+●, 100), 334 (2), 306 (1), 218 (1), 161 (17), 159 (22), 133 (24), 131 (10). FTIR [wa enumbe s (cm−1)]: νN-H: 3,432, 3,340 and 3,229; νC=O: 1,672 and 1,598. In a 25 mL lask i ed wi h a e lux condense and unde N2 a mosphe e, (3Z,6Z)-3-(4- (dime hylamino)benzylidene)-6-(4-aminobenzylidene) pipe azine-2,5-dione (1.2 g, 3.45 mmol) was dissol ed in NMP (7 mL). Me hac yloyl chlo ide (0.47 g, 4.5 mmol) was added o he solu ion, and he mix u e was s i ed a oom empe a u e o 4 h. An o ange solid (monome 3) was collec ed by il a ion and pu i ied om he c ude esidue by washing wi h ho ace one using a Soxhle appa a us. Yield: 80%. M.p.: 330 °C. 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 10.08 (2H, s, NH); 9.97 (1H, s, NH); 7.79 (2H, d, J 8.7, A H); 7.56 (2H, d, J 8.7, A H); 7.48 (2H, d, J 8.7, A H); 6.77 (4H, m, A H); 5.86 (1H, s, CH2); 5.58 (1H, s, CH2); 3.01 (6H, s, CH3); 1.99 (3H, s, CH3). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 167.77, 159.55, 159.02, 151.09, 141.26, 139.89, 131.89, 130.74, 129.22, 126.63, 123.43, 121.44, 121.14, 120.91, 117.64, 115.12, 112.91, 19.80. EI-LRMS m/z: 416 (M+●, 100), 376 (16), 347 (4), 159 (62), 131 (9), 117 (5), 77 (3). FTIR [wa enumbe s (cm−1)]: νN-H: b oadband (3,709, 3,100); νC=O: 1,676, 1,624 and 1,595. Scheme 2. Syn hesis o he monome N-(4-((1Z)-((Z)-5-(4-(dime hylamino)benzylidene)- 3,6-dioxopipe azin-2-ylidene)me hyl)phenyl)me hac ylamide. 2.4. Memb ane P epa a ion Memb ane M1 was p epa ed by he adical polyme isa ion o a mix u e o 1- inyl-2-py olidone and (3) wi h a mola a io o 99.75:0.25. E hylene glycol dime hac yla e was used as he c oss-linking agen (7% mol pe cen age ega ding he o e all comonome mola con en ), and AIBN (1 w %) was used as a he mal adical ini ia o . Memb ane M2 was p epa ed ollowing he same p ocedu e desc ibed o he p epa a ion o M1; howe e , 0.25% mola con en o me cu y(II) ace a e was added ( he same concen a ion o (3)), which esul ed in a hyb id o ganic-ino ganic ma e ial. The he mal polyme isa ion was pe o med in 100 μm hick silanised glass moulds in an oxygen- ee a mosphe e a 65 °C o 5 h. The s uc u e and he physical appea ance a e depic ed in Figu e 1. COOHH2Ni) E hylene glycol, Δ ii) Ac2O, Δ N NO O O O N N H H O O NO2 N i) Na2S.9H2O, Dioxane, Δ ii) , NMP Cl ONNHH O ON N O H (1) (3) (2) i) , BuO-K+, DMF NO 2 OHC NOHC ii) , TEA, DMF, Δ Senso s 2012, 12 2974 Figu e 1. Chemical s uc u es o he monome s and he copolyme . The copolyme is shown o e a digi al pic u e o he senso y ilm. 3. Resul s and Discussion 3.1. Ma e ial Cha ac e isa ion Mechanical and he mal esis ance a e key pa ame e s o de e mine he sui abili y o an o ganic ma e ial o echnological applica ions. F om a mechanical poin o iew, M1, a dense memb ane, showed good pe o mance. The Young’s modulus was 490 MPa and he elonga ion a b eak was 160% a oom empe a u e wi h a ela i e humidi y o 65%. The hyd ophilic memb anes we e d ied a 103 °C o 20 minu es, which esul ed in an inc ease in he Young´s modulus o 1.1 GPa and a dec ease in he elonga ion a b eak o 12%. The memb ane eco e ed he ini ial alues upon exposu e o he ambien a mosphe e. The hyd ophilic cha ac e o he ma e ial esul ed in a wa e up ake o 150% upon imme sing he memb ane in pu e wa e . A compa ison be ween he FTIR spec a o d y M1 and M1 s o ed in ai o e nigh (unde he abo emen ioned condi ions) was pe o med. The band co esponding o he amide I o he hyd ophilic inylpy olidone moie ies exhibi ed a band shi owa d lowe ene gy o 18 cm−1 (1,668 o 1,648 cm−1), while he shoulde a 1,727 cm−1 ha co esponds o he hyd ophobic es e esidue o he c osslinke emained unchanged. These obse a ions p obably indica e ha hyd ophilic and hyd ophobic mic odomains we e p esen in he wa e -swelled memb ane a e imme sing he memb ane in aqueous media o sensing pu poses. The polyme isa ion o he comonome s wi hou and wi h low mola con en me cu y (II) ace a e (0.25%) (M1 and M2, espec i ely) esul ed in ma e ials wi h ai ly di e en FTIR spec a (Figu e 2). Compa ing he spec a o d y samples o M1 and M2 esul ed in he obse a ion o an in ense band ha de eloped a 1,722 cm−1 o M2, wi h a concomi an shi o he amide I band o highe ene gies, om 1,668 o 1,675 cm−1, p obably due o he ace a e g oup. monome s R1R2 (3) N N H H O O N N HO NO R1:R 2: OO O O c osslinke ilm s uc u e monome mola a io: M1: X/Y/Z/M = 99.75/0.25/7.00/0.00 M2: X/Y/Z/M = 99.75/0.25/7.00/0.25 Z R2 O R1 O O O O [Hg(II) ] XY M Senso s 2012, 12 2975 Figu e 2. FTIR spec a o he d ied memb anes M1 (black line) and M2 ( ed line). The he mal esis ance o he memb anes was e alua ed using TGA. The decomposi ion empe a u es ha esul ed in 5% and 10% weigh loss unde a ni ogen a mosphe e (T5 and T10, espec i ely) we e app oxima ely 360 and 385 °C, which indica es he ma e ial had easonably good he mal s abili y. M1 and M2 had a i s weigh loss a 200 °C, which was a ibu ed o he non- e icula ed chain ends [29]. The TGA cu es o he memb anes a e shown in Figu e 3. The esidue emaining a e eaching 800 °C was negligible o M1 and app oxima ely 8% o M2, which con i ms he in luence o he me cu y con en in he he mal beha iou . The me cu y was i s oxidised o HgO, which indica es he hyb id na u e o he memb ane. The imme sion o memb ane M2 in wa e esul ed in an insigni ican loss o bound Hg(II), as de e mined by compa ing he amoun o esidue ha emained a 800 °C unde a ni ogen a mosphe e o wo samples o M2 ha we e soaked in pu e wa e o 3 and 24 h and subsequen ly d ied. Bo h samples esul ed in a esidue o 8%. Ne e heless, he analysis o he ole o he Hg(II) by TGA is cumbe some, because o he beha iou o he me cu y sal s upon hea ing. Ini ially, me cu y oxides o med, and hen, me allic me cu y was o med wi h concomi an sublima ion [30]. Changing he a mosphe e om ni ogen o ai yielded he comple e loss o mass a 800 °C o M1 and M2, which ga e ise o a ze o cha yield. Figu e 3. TGA cu es o memb anes M1 and M2. The deg ada ion pa e n o M2 a e a cycle o soaking in pu e wa e o 3 h wi h subsequen d ying a is also included. 4000 3500 3000 2500 2000 1500 1000 50 0 70 75 80 85 90 95 100 νC=O (lac am, amide I) T ansmi ance (%) Wa enumbe s (cm-1) no malized νC=O (es e , c osslinke ) 100 200 300 400 500 600 700 800 0 20 40 60 80 100 Weigh (%) Tempe a u e (ºC) M2 M2 in wa e (3h) M1 Senso s 2012, 12 2976 3.2. The Memb anes as Senso y Ma e ials The memb ane M1 beha es as a senso y ma e ial o he luo ogenic de ec ion o Hg(II) in aqueous media. Upon he addi ion o Hg(II), he luo escence o he memb ane a 548 nm was quenched, which demons a ed ha he memb ane had “ u n-o ” luo escence beha iou in he p esence o he ca ion. This obse a ion was a ibu ed o he in e ac ion o he Hg(II) wi h he N- e minus o he senso y mo i (3) wi hin he memb ane a a 1:1 s oichiome y [31]. The in eg al p epa a ion o a memb ane con aining equal mola quan i ies o (3) and Hg(II) (i.e., M2), led o a ma e ial wi h a pa ially quenched luo escence. Mo eo e , luo escence eco e y was obse ed o M2 upon adding di e en biomolecules, e.g., CoA, Cys and GSH (see Figu e 4). The s onge in e ac ion o hese biomolecules wi h Hg(II) led o a luo escen chemosenso wi h luo escence “ u n-on” beha iou , based on he displacemen app oach [1,2]. A i a ion cu e o he biomolecules was ob ained by plo ing he luo escence maxima e sus he biomolecule concen a ion. An illus a i e example is shown o CoA in Figu e 4. The limi o de ec ion (LOD) was app oxima ely 2 × 10−10 M. Figu e 4. Selec ed luo escence spec a (le ) and i a ion cu e ( igh ) o M2 upon adding inc easing quan i ies o CoA in wa e a physiological pH (pH = 7.4, TRIS) a an exci a ion wa eleng h o 400 nm. The inse o he igu e on he igh is an expansion o he lowe concen a ions o he i a ion cu e. 3.3. Copolyme Ne wo k/Hg(II) In e ac ion P io o he p epa a ion o he memb ane M2, he in e ac ion o he monome con aining he sensing mo i (3) wi h Hg(II) was s udied in solu ion. The s oichiome y o he (3):Hg(II) complexes in a DMSO/wa e solu ion (90:10) was ma hema ically de e mined by analysing he luo escence quenching p ocess, he in ensi y maxima a ia ions e sus he Hg(II) concen a ion, and he co esponding Job’s plo s. The Job’s plo showed a maximum ha appea ed a a mole ac ion o (3) (χ(3)) o 0.5, which clea ly indica ed he o ma ion o complexes wi h a 1:1 s oichiome y, as shown in Table 1 and Figu e 5. 530 540 550 560 570 580 590 70 72 74 76 78 80 82 84 86 8.2x10-11 M Fluo escence in ensi y (a.u.) [CoA] 6.1x10-8 M λ, nm 0.0 2.0x10-8 4.0x10-8 6.0x10-8 0.96 0.97 0.98 0.99 1.00 0.0 1.0x10-9 2.0x10-9 3.0x10-9 4.0x10-9 0.97 0.98 0.99 1.00 I0/I (549 nm) [CoA], M I0/I (549 nm) [CoA], M Senso s 2012, 12 2977 Table 1. S abili y cons an s co esponding o he complex (3):X [X = Hg(II), CoA, Cys and GSH], and CoA:Hg(II). Complex Complex s oichiome y K1 (M−1) K2 (M−1) (3):Hg(II) 1:1 110,000 ± 10,000 – (3):CoA 1:1 22,000 ± 2,000 – (3):Cys 1:1 20,000 ± 8,000 – (3):GSH 1:1 20,000 ± 7,000 – CoA:Hg(II) 1:2 8,400 ± 900 6,000 ± 3,000 Figu e 5. Job’s plo s co esponding o he ollowing in e ac ion s udies: (a) (3) wi h Hg(II), ob ained om luo escence spec oscopy (591 nm) da a co esponding o he i a ion cu e o (3) wi h me cu y ca ions in DMSO/wa e (90/10, / ) a pH = 9.7 (TRIS); and (b) CoA wi h Hg(II), om UV/Vis spec oscopy (300 nm) da a co esponding o he i a ion o CoA wi h me cu y ca ions in DMSO/wa e (90/10, / ) a pH = 7.4 (TRIS). (a) (b) The s eng h o he in e ac ion o (3) wi h Hg(II) in bu e ed DMSO/wa e (90/10) solu ion (pH = 7.4) in e ms o he s abili y cons an , K1, co esponding o he hos -gues complexes, was analysed by luo escence spec oscopy. The de e mina ion o he 1:1 s oichiome y o he (3):Hg(II) complexes allowed he ollowing equilib ium o be s a ed: L + M ML K1 (1) which can also be w i en as he ollowing: ]][[ ][ 1ML ML K= (2) whe e [L], [M], and [ML] is he equilib ium concen a ion o (3), Hg(II), and he (3):Hg(II) complex, espec i ely. F om he mass balance and luo escence da a, he ollowing equa ions can be deduced: CL = [L] + [ML] (3) CM = [M] + [ML] (4) IF = L[L] + ML[ML] (5) 0.0 0.2 0.4 0.6 0.8 1.0 0 10 20 30 40 (Fo-F)χ(3) χ(3) 0.0 0.2 0.4 0.6 0.8 1.0 0.00 0.05 0.10 0.15 (A-Ao)χCoA χCoA