1566 DOI: 10.1021/la904138f Langmuir 2010, 26(3), 1566–1569Published on Web 01/05/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Colorimetric Sensing of r-Amino Acids and Its Application for the

“Label-Free” Detection of Protease

Xiaoding Lou,† Liyao Zhang,*,‡ Jingui Qin,† and Zhen Li*,†

†Department of Chemistry, Hubei Key Lab on Organic and Polymeric Opto-Electronic Materials and‡College of Life Science, Wuhan University, Wuhan 430072, China

Received November 3, 2009. Revised Manuscript Received December 22, 2009

Anew indirect approach to explore sensitive colorimetric sensors towardR-amino acids is proposed: the pink solutionof 1 and copper ions changed to colorless immediately upon the addition of R-amino acids. As the hydrolysis of bovineserum albumin (BSA) with the aid of trypsin produces R-amino acids, the complex of 1/Cu2þ/BSA could act as alabel-free, sensitive, selective sensor toward trypsin. The detection process could be visually observed by naked eyes.

Many efforts have been attracted to the development of newsensors toward biological macromolecules, such as protein andnucleic acids, as the result of increasing attention paid tohuman health, and diagnosis and treatment of disease.1,2 Askey constituents of proteins, R-amino acids are importantanalytes to be detected. Thanks to the enthusiastic efforts ofscientists, many analytical methods have been developed, suchas NMR, UV absorption, fluorescent emission, and variousseparation techniques in the molecular recognition of aminoacids.3,4 However, most of them are laborious, especially the

difficulty in designing materials with suitable structure tointeract with R-amino acids with detectable signals. Similarcases are present in sensors toward anions.5 On the contrary,sensors toward metal ions could be easily explored, as we knowmuch about the interactions between ligands and metal ions.Thus, it might be a wonderful thing if we could develop newsensors for anions,R-amino acids, and other neutral species, bythe utilization of the rules for metal ion sensors. This is reallypossible, since many of these species can form stable complexeswith cations, and might snatch cations from the formedcomplex of the cations and their corresponding sensors, witha detectable signal, if the stability constant of the complexformed by the analyte and the cation is larger than that of thecomplex of the cation and its sensor. As to R-amino acids, theycould form stable complexes with some metal ions, that is,copper ions (Chart 1).6 Therefore, it should be a feasibleapproach to find new R-amino acid sensors from those forcopper ions. Our previous example confirmed this point: thestrong fluorescence of the conjugated polymers could bequenched by trace copper ions, and upon the addition ofR-amino acids the quenched fluorescence would recover, mak-ing these conjugated polymers new chemsensors towardR-amino acids.7 However, considering that colorimetric che-mosensors are widely used owing to the low cost and lack ofequipment required, and a color change can be easily observedby the naked eyes, even at very low analyte concentrations, wewould like to develop new colorimetric R-amino acid sensorsaccording to the above idea. To the best of our knowledge, sofar, there are no reports about this idea.

As we know, there are many good chemosensors for copperions with high sensitivity and selectivity, including colorimetricones. In this Letter, we would like to report the sensing behavior

Chart 1

*To whom correspondence should be addressed. Fax: þ86-27-6875-6757.Telephone: þ86-27-6225-4108. E-mail: lizhen@whu.edu.cn (Z.L.); lyzhang26@yahoo.com.cn (L.Z.).(1) (a) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107,

1339. (b) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.;McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515. (c) Chen, L.;McBranch, D. W.; Wang, H.-L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl.Acad. Sci. U.S.A. 1999, 96, 12287. (d) Li, C.; Numata, M.; Takeuchi, M.; Shinkai, S.Angew. Chem., Int. Ed. 2005, 44, 6371.(2) (a) Ho, H.-A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384. (b) Gaylord, B.

S.; Wang, S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2001, 123, 6417. (c)Folmer-Andersen, J. F.; Lynch, V. M.; Anslyn, E. V. Chem.;Eur. J. 2005, 11, 5319.(d) He, F.; Tang, Y.; Yu, M.; Wang, S.; Li, Y.; Zhu, D. Adv. Funct. Mater. 2006, 16, 91.(e) Miranda, O. R.; You, C. C.; Phillips, R.; Kim, I. B.; Ghosh, P. S.; Bunz, U. H. F.;Rotello, V. M. J. Am. Chem. Soc. 2007, 129, 9856. (f) Andersen, J. F. F.; Lynch, V. M.;Anslyn, E. V. J. Am. Chem. Soc. 2005, 127, 7986.(3) (a) Ryu, D.; Park, E.; Kim, D. S.; Yan, S.; Lee, J. Y.; Chang, B. Y.; Ahn, K.

H. J. Am. Chem. Soc. 2008, 130, 2394. (b) Feuster, E. K.; Glass, T. E. J. Am. Chem.Soc. 2003, 125, 16174. (c) Czarnik, A. W. Chem. Biol. 1995, 2, 423. (d) Mizutani, T.;Wada, K.; Kitagawa, S. J. Am. Chem. Soc. 1999, 121, 11425. (e) At-Haddou, H.;Wiskur, S. L.; Lynch, V. M.; Anslyn, E. V. J. Am. Chem. Soc. 2001, 123, 11296. (f)Zhang, M.; Yu, M.; Li, F.; Zhu, M.; Li, M.; Gao, Y.; Li, L.; Liu, Z.; Zhang, J.; Zhang, D.;Yi, T.; Huang, C. J. Am. Chem. Soc. 2007, 129, 10322. (g) Tundo, P.; Fendler, J. H.J. Am. Chem. Soc. 1980, 102, 1760. (h) Lehn, J. M. Angew. Chem., Int. Ed. Engl.1988, 27, 89. (i) Leipert, D.; Nopper, D.; Bauser, M.; Gauglitz, G.; Jung, G. Angew.Chem., Int. Ed. Engl. 1998, 37, 3308. (j) Sasaki, S.; Hashizume, A.; Citterio, D.; Fujii,E.; Suzuki, K. Angew. Chem., Int. Ed. Engl. 2002, 41, 3005.(4) (a) Folmer-Andersen, J. F.; Lynch, V. M.; Anslyn, E. V. Chem.;Eur. J.

2005, 11, 5319. (b) Imai, H.; Munakata, M.; Uemori, Y.; Sakura, N. Inorg. Chem. 2004,43, 1211. (c) Fabbrizzi, L.; Licchelli, M.; Parotti, A.; Poggi, A.; Rabaioli, G.; Sacchi, D.;Taglietti, A. J. Chem. Soc., Perkin Trans. 2 2001, 11, 2108. (d) Kruppa, M.; Mandl, C.;Miltschitzky, S.; Knig, B. J. Am.Chem. Soc. 2005, 127, 3362. (e) Schmuck, C.; Bickert,V.Org. Lett. 2003, 5, 4579. (f) Severin, K.; Bergs, R.; Beck,W.Angew. Chem., Int. Ed.1998, 37, 1634. (g) Gerley, A.; Sovago, I.; Nagypal, I.; Kiraly, R. Inorg. Chim. Acta1972, 435. (h) Chin, J.; Lee, S. S.; Lee, K. J.; Park, S.; Kim, D. H.Nature 1999, 401, 254.(i) Pagliari, S.; Corrandini, R.; Galaverna, G.; Sforza, S.; Dossena, A.; Montalti, M.;Prodi, L.; Zaccheroni, N.; Marchelli, R. Chem.;Eur. J. 2004, 10, 2749. (j) Hortala,M. A.; Fabbrizzi, L.; Marcitte, N.; Stomeo, F.; Taglietti, A. J. Am. Chem. Soc. 2003,125, 20. (k) Buryak, A.; Severin, K. Angew. Chem., Int. Ed. 2004, 43, 4771.(5) (a) Gale, P. A.Coord. Chem. Rev. 2000, 199, 181. (b) Gale, P. A.Coord. Chem.

Rev. 2001, 213, 79. (c) Sessler, J. L.; Davis, J. M. Acc. Chem. Res. 2001, 34, 989.(d) Lee, D. H.; Kim, S. Y.; Hong, J. I. Angew. Chem., Int. Ed. 2004, 43, 4777. (e)Martínez-M�a~nez, R.; Sancen�on, F. Chem. Rev. 2003, 103, 4419. (f) Schmidtchen, F. P.;Berger, M. Chem. Rev. 1997, 97, 1515.

(6) (a) Xing, Q.; Xu, R.; Zhou, Z.; Pei, W. Organic Chemistry, 2nd ed.; HigherEducation Press: Beijing, China, 1994;(b) Everin, K.; Bergs, R.; Beck, W. Angew.Chem., Int. Ed. Engl. 1998, 37, 1635.

(7) (a) Zeng, Q.; Zhang, L.; Li, Z.; Qin, J.; Tang, B. Z. Polymer 2009, 50, 434. (b)Li, Z.; Lou, X.; Li, Z.; Qin, J. ACS Appl. Mater. Interfaces 2009, 1, 232.

DOI: 10.1021/la904138f 1567Langmuir 2010, 26(3), 1566–1569

Lou et al. Letter

of a derivative of Rhodamine B (compound 1), a good copper ionchemosensor,8 toward R-amino acids. The obtained experimentalresults demonstrated that, with the aid of copper ions, compound1 was really a highly sensitive and selective sensor for R-aminoacids. Not just satisfied with the results, considering thatR-aminoacids could be produced in the hydrolysis process of protein in thepresence of protease, moreover, we have also successfully appliedthe new R-amino acid sensor to detect protease selectively andsensitively, with the assay strategy demonstrated in Scheme 1.

Compound 1, with its structure shown in Scheme 1, was easilyprepared according to the literature.8 Also, we could follow theoptimized testing conditions of compound 1 toward copper ions.Really, upon the addition of copper ions, the original colorlesssolution of compound 1 became pink soon and the absorption at555 nm was as high as about 0.5, when the concentrations ofcompound 1 and copper ions were 1� 10-5 and 7� 10-6 mol/L,respectively. Then we tried to add some R-amino acids, forexample, alanine (Ala), to the complex of 1 and copper ions,and immediately the pink solution became colorless step by step:when the concentration of Ala was 2 � 10-4 mol/L, the pinksolution completely faded into colorless (Figure S1, SupportingInformation). All of the other R-amino acids (their structuresshown in Chart S1, Supporting Information) were utilized tocheck the sensing properties of the complex of 1 and copper ions,instead of Ala. As shown in Figures S2-S16 in the SupportingInformation, the complex of 1 and copper ions could give a quickresponse to theseR-amino acids, especially toward histidine (His):at the concentration of His as low as 1.3 � 10-5 mol/L, thesolution was colorless. This is reasonable that the detectionsensitivity of His was higher than that of other R-amino acids(Figure 1), since the imidazolemoieties in it (Chart S1, SupportingInformation) might help His snatch the copper ions from thecomplex of 1 and copper ions.

As mentioned above, one of the advantages of colorimetricsensors is that the color change could be seen by naked eyes. Asdemonstrated in Figure 2, when the concentration was 2 � 10-4

mol/L, the solution color of the complex of 1 and copper ionschanged from pink to colorless upon the addition of any one ofthe R-amino acids, indicating that, by using the indirect methodreported here, R-amino acids could be detected visually.

Considering there were generally three isomers of amino acids,R-, β- and γ-amino acids, we wanted to determine if 1 could givedifferent responses to them. Accordingly, R-, β- and γ-amino-butyric acids (ABAs) (Chart S2, Supporting Information) wereused to evaluate the selectivity of 1 toward R-amino acids. Asshown in Figure 3, while the addition of R-ABA faded the pinksolution of the complex of 1 and copper ions into colorless, thesolution of the 1/Cu2þ complex remained light pink upon theaddition of β- and γ-ABAs. Their UV-vis spectra gave more

accurate data (Figures S17-S20, Supporting Information). Themain reason might be that the complexes of R-amino acids andcopper ions were more stable with the formed five-member ringsthan those fromβ- andγ-amino acids. These results demonstratedthat 1 could be considered as a good selective sensor for R-aminoacids. Thus, combined with the high sensitivity, the example of 1realized our thought of developing new colorimetricR-amino acidchemosensors through an indirect approach.

Although the obtained experimental results have proved theindirect trick, and compound 1 could be utilized to probe the traceR-amino acids in some special cases when needed, we still wantedto further explore all of the potential applications of the 1/Cu2þ

complex. It is well-known that R-amino acids can be produced inthe hydrolysis process of protein in the presence of protease, andthus, this point could be applied to probe the presence of specificprotease, since the produced R-amino acids can be easily detectedby our method reported above and protease can only hydrolyzethe corresponding protein selectively.

As reported in the literature, trypsin is the most importantdigestive enzyme produced by the pancreas and plays a key role incontrolling pancreatic exocrine function. The trypsin level isincreased with some types of pancreatic diseases such as acutepancreatitis and cystic fibrosis.9 To assay trypsin activity, differ-ent methods have been employed.10 Based on the good sensingperformance of 1 toward R-amino acids, our assay approach fortrypsin is shown in Scheme 1, since as a member of the serineprotease family trypsin can cleave peptides on the C-terminal sideof lysine and arginine amino acid residues to produce amino acidor peptide fragments with stronger chelating ability to copperions. Accordingly, a solution of the 1/Cu2þ complex and bovineserum albumin (BSA) as substrate of protease was prepared;upon the addition of protease, the substrate protein would be

Scheme 1. Schematic Representations of the Detection for Trypsin

Figure 1. UV-vis spectra of 1/Cu2þ/different R-amino acid. Theconcentration of each R-amino acid was 1.3 � 10-5 mol/L. Theconcentration of 1 was 1.0 � 10-5 mol/L, while that of Cu2þ was7.0 � 10-6 mol/L.

(8) Xiang, Y.; Tong, A.; Jin, P.; Ju, Y. Org. Lett. 2006, 8, 2863.

(9) Ionescu, R. E.; Cosnier, S.; Marks, R. S. Anal. Chem. 2006, 78, 6327.(10) (a) Wosnick, J. H.; Mello, C. M.; Swager, T. M. J. Am. Chem. Soc. 2005,

127, 3400. (b) Josephson, L.; Kircher, M. F.; Mahmood, U.; Tang, Y.; Weissleder, R.Bioconjugate Chem. 2002, 13, 554. (c) Kircher, M. F.; Weissleder, R.; Josephson, L.Bioconjugate Chem. 2004, 15, 242. (d) An, L.; Liu, L.; Wang, S. Biomacromolecules2009, 10, 454. (e) An, L.; Tang, Y.; Feng, F.; He, F.;Wang, S. J.Mater. Chem. 2007, 17,4147. (f) Ingram, A.; Byers, L.; Faulds, K.; Moore, B. D.; Graham, D. J. Am. Chem.Soc. 2008, 130, 11846.

1568 DOI: 10.1021/la904138f Langmuir 2010, 26(3), 1566–1569

Letter Lou et al.

cleaved into amino acids or peptide fragments, which couldsnatch the copper ions from the complex of 1 and copper ions,accompanying the detectable optical signal, here a color change,whichmight beobserved bynaked eyes. Since therewas 50%(v/v)water in the above sensing system toward R-amino acids, thedetection of the protease activity might be conducted in real time.To exclude the possible inherent influence of BSA, controlexperiments were designed for comparison. As shown in FiguresS21-S23 in the Supporting Information, there was nearly noinfluence fromBSA, regardless of the concentration ofBSAbeingas high as 20 μg/mL or the test time being as long as 40 min. Thiswas reasonable, since in global BSA there is only one specificbinding site for copper ions,11 and this interaction is too weak tograb copper ions from its complex with 1.

In a typical experiment, a solution containing 1.0� 10-5mol/Lof 1, 7.0 � 10-6 mol/L of Cu2þ, and 10 μg/mL of BSA wasprepared in Tris-HCl (10 mM, pH= 7.0) buffer containing 50%(v/v) water/CH3CN at 32 �C. After the addition of some trypsin,theUV-vis intensity of 1/Cu2þ/BSAwasmonitored as a functionof time. As shown in Figures S24 and S25 in the SupportingInformation, the UV-vis spectra were measured at 2 min inter-vals over 20 min. The UV/vis spectrum of 1/Cu2þ/BSA solutionwas characterized by a very intense band centered at 555 nm,which was responsible for the magenta color of the solution.Upon the addition of trypsin (3.0 μg/mL), the intensity of theabsorption maximum of 1/Cu2þ/BSA at 555 nm graduallydecreased with incubation time from 0 to 20 min and remainednearly unchanged after 18 min, indicating that the digestion ofBSA was almost complete. This disclosed that the 1/Cu2þ/BSAcomplex can be utilized as a probe to detect trypsin in real time.

To further check the sensing phenomena of trypsin, wechanged its concentration in the testing system. Figure 4 demon-strates the dependence of the intensity ratio (A - A0) of 1 at555 nm on the concentration of trypsin (0 5.0 μg/mL) with fixedconcentrations of 1, copper ions, and BSA. A nearly linear

relationship between the maximum absorption intensity and theconcentration of trypsin was observed over the enzyme from 0 to5.0 μg/mL, indicating that the 1/Cu2þ/BSA complex could assaythe trypsin quantitatively. The increase in the trypsin amountgave rise to a fast initial cleavage reaction rate and a higher level ofUV-vis spectra response of complex 1 at the same initialconcentration of BSA. And the cleavage of BSA by trypsinresulted in the absorbance change (A - A0) as big as 0.28,demonstrating the good UV-vis spectra response of the1/Cu2þ/BSA complex toward trypsin (Figure 4 and FiguresS24-S32, Supporting Information). Also, the hydrolysis processofBSA in the presence of trypsin could be visually observedby thenaked eyes, just as shown in Figure 5.

Figure 2. Different solutions of 1 and copper ions in Tris-HCl (10 mM, pH= 7.0) buffer containing 50% (v/v) water/CH3CN (1.0� 10-5

and 7.0� 10-6mol/L, respectively) in the presence of differentR-amino acids (2.0� 10-4mol/L). From left to right: 1, 1þCu2þ, 1þCu2þþAla, Pro, Gly, Cit, Met, Phe, Val, Leu, Ser, Thr, Asn, Ile, Arg, Lys, Trp, HA, and His.

Figure 3. Different solutions of 1 and copper ions in Tris-HCl(10 mM, pH = 7.0) buffer containing 50% (v/v) water/CH3CN(1.0� 10-5 and 7.0 � 10-6 mol/L, respectively) in the presence ofdifferentABAs (2.0� 10-4mol/L). From left to right: 1, 1þCu2þ,1þ Cu2þþR-ABA, 1þ Cu2þ þ β-ABA, and 1þ Cu2þ þ γ-ABA.

Figure 4. Absorption difference at λ= 555 nm on the concentra-tionof trypsinwith fixed concentrations of1, copper ions, andBSAinTris-HCl (10mM,pH=7.0) buffer containing50%(v/v)water/CH3CN. The concentration of 1 was 1.0 � 10-5 mol/L, [Cu2þ] =7.0� 10-6mol/L, [BSA]=10μg/mL, and [trypsin]=05.0μg/mL.

Figure 5. Different solutions of 1, copper ions, and BSA in Tris-HCl (10 mM,pH = 7.0) buffer containing 50% (v/v) water/CH3CN (1.0� 10-5, 7.0� 10-6mol/L and 10μg/mL, respectively)in the presence of trypsin (3.0 μg/mL). From left to right: 1, 1 þCu2þ, and 1 þ Cu2þ þ BSA þ trypsin (0, 5, 10, 15, and 20 min).

(11) Filenko, A.; Demchenko, M.; Mustafaeva, Z.; Osada, Y.; Mustafaev, M.Biomacromolecules 2001, 2, 270.

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Lou et al. Letter

To evaluate the selectivity of substrates on the assay of trypsin,three other proteins, including Con A, papain, and lysozyme assubstrates, were studied instead of BSA at the same experimentalconditions presented in Figure 6 and Figures S33-S35 in theSupporting Information. The results demonstrated that it wasonlyBSAwhich could be hydrolyzedby trypsin efficiently. On theother hand, the UV-vis responses of 1/Cu2þ/BSA were alsostudied in the presence of a nonspecific enzyme, pepsin. As shownin Figures S36 and S37 in the Supporting Information, there wasno big difference before and after the addition of pepsin, indicat-ing that BSA could not be hydrolyzed by pepsin. Therefore, the1/Cu2þ/BSA complex could be utilized to sensitively detecttrypsin with minor interference from other nonspecific substrates

and proteases. Or in other words, the usage of BSA as substrate initself determined the selective detecting behavior of the resultantsensing system of the 1/Cu2þ/BSA complex toward trypsin. Thus,from this point, it is reasonable to deduce that, just by using otherprotein instead of BSA as substrate, other corresponding pro-teases could be selectively probed. So, the example presented inthis Letter opens up a new avenue to develop new colorimetricsensors toward different proteases, in addition to the detection ofR-amino acids.

In conclusion, we have successfully developed a “novel” sensortoward R-amino acids based on a good copper ion chemosensor.The preliminary results demonstrated the following: (1) For thefirst time, the reported colorimetric copper ion chemosensor wasused to sense R-amino acids, sensitively and selectively, byutilizing the different stability constants of copper ions towardcompound 1 and R-amino acids. The good sensing performanceof compound 1 was successfully applied to the “label-free”detection of protease, sensitively and selectively. It is believedthat more sensors could be developed through this indirectstrategy. (2) The good selectivity and high sensitivity of com-pound 1 toward R-amino acids and protease, coupled with visualdetection by the naked eyes, made compound 1 a promisingcandidate for practical applications as a good bioprobe.

Acknowledgment. We are grateful to the National ScienceFoundation of China (Nos. 20974084, 30800086) and the pro-gram of NCET for financial support.

Supporting Information Available: Details of the experi-mental procedure; UV-vis spectra; and charts of stuctures.This material is available free of charge via the Internet athttp://pubs.acs.org.

Figure 6. UV-vis spectra of 1/Cu2þ/protein before and afterincubation with trypsin in Tris-HCl (10 mM, pH = 7.0) buffercontaining 50% (v/v) water/CH3CN. The concentration of 1 was1.0� 10-5 mol/L, [Cu2þ] = 7.0� 10-6 mol/L, [protein] = 10 μg/mL, and [trypsin] = 3.0 μg/mL.