ChemComm View Article Online


Cite this: Chem. Commun., 2014, 50, 61

Published on 01 November 2013. Downloaded on 21/01/2014 11:34:36.

Received 27th September 2013, Accepted 28th October 2013

View Journal | View Issue

‘‘Turn-on’’ fluorescent sensor array for basic amino acids in water† Tsuyoshi Minami,a Nina A. Esipenko,a Ben Zhang,b Lyle Isaacs*b and Pavel Anzenbacher, Jr.*a

DOI: 10.1039/c3cc47416j

Amino acids and their derivatives are recognized and analyzed in water using a turn-on fluorescent cucurbituril based sensor array. Multivariate analysis (LDA and HCA) clearly shows that the sensor array can discriminate amino acids from the corresponding amines which are produced by the action of amino acid decarboxylases.

Amino acids are the essential components of life processes. Sensing of amino acids is necessary in diverse fields such as nutritional analysis,1 and the diagnosis of Alzheimer disease2 and pancreatitis.3 Amino acid analysis is usually performed by chromatographic4 or electrochemical5 methods. These techniques are relatively expensive and require trained personnel. Hence, methods amenable to high-throughput assays that require lowcost instrumentation are desirable. Supramolecular receptors and chemosensors for detection and recognition of amino acids have been reported.6 To devise supramolecular sensor arrays for amino acids, we focused on cucurbit[n]uril receptors (CB[n]) capable of binding protonated amines via ion–dipole interactions and hydrogen bonding to the CQO moieties of the ureidyl portals.7 Previously, CB[n]s have been used in fluorescent or colourimetric sensors, however, the nonchromophoric nature of these molecules limits their applications to dye displacement assays.8 To the best of our knowledge, ‘‘turn-on’’ fluorescent sensor arrays for detection of amino acids in water have never been reported. To obtain ‘‘turn-on’’ fluorescent sensors for amino acids, we used fluorescent cucurbituril derivatives,9 probes 1 and 2 (Fig. 1). Both probes display cross-reactive10 binding of a number of amines and amino acids. Probe 1 which is derived from the rigid cucurbit[6]uril macrocyclic receptor displays higher affinity for small guests, whereas flexible acyclic receptor 2 binds preferably larger molecules, a

Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, Ohio 43403, USA. E-mail: [email protected]; Fax: +1-419-372-9809; Tel: +1-419-372-2080 b Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA † Electronic supplementary information (ESI) available: Fluorescence titrations, experimental detail of microarray, canonical scores plots and jackknifed classification matrices. See DOI: 10.1039/c3cc47416j

This journal is © The Royal Society of Chemistry 2014

Fig. 1

Structures of guests and probes used in this study.

thus displaying a complementary selectivity, which enables recognition and quantification of structurally varied analytes. The fluorescence signal in both probes originates from the naphthalene fluorophores whose fluorescence is partly quenched by Eu3+ ion coordinated to the CQO moieties.11 This quenching is due to the energy transfer (antenna effect) from the naphthalene moieties to the Eu3+ ion. The Eu3+ luminescence is not, however, observed due to water molecules coordinated to the Eu3+.12 The naphthalene fluorescence is recovered when the analyte displaces the Eu3+ from the probe–Eu3+ complex (‘‘turn-on’’ response). Simultaneously, the fluorescence output of the probe–guest complex is modulated by the structural features of the guest. Because different guests are

Chem. Commun., 2014, 50, 61--63 | 61

View Article Online

Published on 01 November 2013. Downloaded on 21/01/2014 11:34:36.



Fig. 2 Panel A: the electrospray ionization mass spectrum (ESI MS) of the complex of 2 and arginine. Inset: calculated isotope pattern for C68H83N20O26S43 ; B: the ESI MS spectrum of the complex of 2 and lysine. Inset: calculated isotope pattern for C68H83N18O26S43 .

accommodated by probe 1 and 2 with different affinity, the degree of fluorescence recovery is also unique to the guest. Thus, combining both probes in one sensor array results in a high-performance assay capable of recognizing multiple amino acid-related analytes. Because of the potential importance of assays to detect the conversion of an amino acid to amines as well as detection of drugs in biomedical applications we selected the following 10 analytes: ornithine, putrescine, lysine, cadaverine, arginine, agmatine, histidine, histidinol, histamine, and lisinopril (Fig. 1). These are substrate–product pairs for amino acid decarboxylase;13 lisinopril is an angiotensin-converting enzyme (ACE) inhibitor.14 First, the binding of the amino acids to probes was confirmed using a mass spectrometry (Fig. 2). The MS data showed a 1 : 1 binding mode between the probe and the amino acid. Furthermore, fluorescence titration experiments also confirmed the binding of amino acids to probes. Fig. 3A illustrates the fluorescence spectra recorded for a 1–Eu3+ complex upon titration with a lysine solution at pH 7. Here, the fluorescence increases with the increase of lysine concentration. The fluorescence titration of 2 by arginine showed also a fluorescence enhancement (Fig. 3B). Table 1 displays apparent affinity constants (Kassoc) for the selected amino acids and the corresponding amines. The affinity constants confirm the cross-reactive response as well as size complementarity presumed for probes 1 and 2. Thus, the binding affinity of 2 for a large lisinopril molecule is five times higher than that for 1. The binding constant of 1 for a smaller ornithine is significantly higher than that for 2. This is due to the difference of the probe cavity sizes. Also, one can observe a trend in affinity constants based on the molecular structures of the guests. For example, the magnitude of binding affinities for histidine and the related compounds follows the order histamine > histidinol > histidine. The presence of a carboxyl group lowers the binding affinity presumably due to electrostatic repulsion between the ureidyl CQO moieties of the probes and the carboxy oxygens of amino acids. The difference in binding affinities is expected to contribute to discrimination of amino acids from the corresponding amines in an array-based assay.

62 | Chem. Commun., 2014, 50, 61--63

Fig. 3 (A) Fluorescence spectra of 1 (3 mM) with Eu3+ (300 mM) upon the addition of lysine in water at pH 7. lex = 266 nm. (B) Spectra of 2 (3 mM) upon the addition of arginine in water at pH 7. lex = 301 nm. All probes were excited at the isosbestic point of their absorption or at the point where no change in absorption is observed.

To test this hypothesis the responses of probes were recorded as fluorescence intensities at 320 and 370 nm from probe–Eu3+–guest solutions using conventional 1536-well microplates (see the ESI† for a detailed description). The response data sets were acquired in the form of a guest (X)  [Eu3+] (Y)  lEm (Z) (probe, metal, emission wavelength) matrix, each XiYi Zi field being associated with a various degree of fluorescence enhancement. Pattern recognition protocols were then used to reveal the guest-specific trends in the overall response. Linear discriminant analysis (LDA)15 using leave-one-out crossvalidation was performed. LDA demonstrated that the array can clearly discriminate 10 analytes and a control. Fig. 4 describes the response space defined by the first three canonical factors (F1–F3), showing that 95% of variance is enough to discriminate the analytes. The Jackknife cross-validation yields 100% correct classification of all 220 data-points (corresponding to 10 analytes and control). Furthermore, hierarchical clustering analysis (HCA)15 was carried out. As a significant difference between LDA and HCA, HCA does not reduce dimensions of the datasets, in other words, obtained distances between clusters in HCA correspond to similarities in behaviour of the original samples. Importantly, the HCA dendrogram

This journal is © The Royal Society of Chemistry 2014

View Article Online

ChemComm Table 1


The apparent association constants (Kassoc, M 1)a obtained from fluorescence titration

Guest Host 1 2

Ornithine 2

9.9  10 NDc

Putrescine 6

>10 1.9  104


Cadaverine 3

1.6  10 3.4  102


>10 >106




1.8  10 1.5  103


>10 >106

Histidine 3

1.7  10 NDc

Histidinol 3

2.9  10 1.5  103

Histamine 4

1.7  10 6.4  103

Lisinoprilb 7.2  102 3.8  103

Published on 01 November 2013. Downloaded on 21/01/2014 11:34:36.

a The titrations recorded in the presence of Eu3+ (300 mM) and Kassocs were calculated based on the change in fluorescence intensity upon addition of each guest in water at pH 7. The errors of the curve fitting were o20%. b The titration was carried out in water at pH 3 to increase the solubility. c Kassoc could not be calculated due to the insufficient change in fluorescence response.

In summary, we demonstrated a simple yet reliable microarray approach utilising two complementary cucurbit[n]uril-type receptors to discriminate basic amino acids from the corresponding amines in water. The qualitative analysis of 10 biologically relevant analytes by LDA and HCA yield 100% correct classification, which confirms the excellent guest-recognition properties of the probes. To the best of our knowledge, this is the first report of a turn-on fluorescent microarray utilizing directly fluorescent sensors for amino acids in water. We believe that these results open up an avenue for development of future array sensors for detection of amino acids in biomedical applications. L.I. acknowledges support from NSF (CHE-1110911); P.A. acknowledges support from NSF (CHE-0750303 and DMR-1006761).


Fig. 4 LDA canonical score plot for the response of the sensors array to ten analytes (and control) in water. The cross-validation routine shows 100% correct classification. For more details see the ESI.†

Fig. 5 Hierarchical clustering analysis (HCA) dendrogram (Ward linkages, Pearson distances) display s the analysis of all 11 classes (total of 220 trials) and perfect 100% correct classification.

(Fig. 5) displayed the 100% correct classification. In the dendrogram, two main analyte clusters are distinguished: amines on the right side and amino acids on the left side. This shows that the sensor array can discriminate amino acids from the corresponding products of amino acid decarboxylation.

This journal is © The Royal Society of Chemistry 2014

1 M. Freidman, J. Agric. Food Chem., 1999, 47, 3457. 2 A. D’Aniello, A. Vetere, G. H. Fisher, G. Cusano, M. Chavez and L. Petrucelli, Brain Res., 1992, 592, 44. 3 R. E. Ionescu, S. Cosnier and R. S. Marks, Anal. Chem., 2006, 78, 6327. 4 J. Le Boucher, C. Charret, C. Coudray-Lucas, J. Giboudeau and L. Cynober, Clin. Chem., 1997, 43, 1421. 5 G. L. Luque, N. F. Ferreyra and G. A. Rivas, Talanta, 2007, 71, 1282. 6 L. Fabbrizzi, M. Licchelli, G. Rabaioli and A. Taglietti, Coord. Chem. Rev., 2000, 205, 85. 7 J. Lagona, P. Mukhopadhyay, S. Chakrabarti and L. Isaacs, Angew. Chem., Int. Ed., 2005, 44, 4844. 8 (a) L. A. Baumes, M. Buaki, J. Jolly, A. Corma and H. Garcia, Tetrahedron Lett., 2011, 52, 1418; (b) A. Hennig, H. Bakirci and W. M. Nau, Nat. Methods, 2007, 4, 629; (c) D. M. Bailey, A. Hennig, V. D. Uzunova and W. M. Nau, Chem.–Eur. J., 2008, 14, 6069; (d) J. Lagona, B. D. Wagner and L. Isaacs, J. Org. Chem., 2006, 71, 1181. 9 (a) D. Lucas, T. Minami, G. Iannuzzi, L. Cao, J. B. Wittenberg, P. Anzenbacher, Jr. and L. Isaacs, J. Am. Chem. Soc., 2011, 133, 17966; (b) T. Minami, N. A. Esipenko, B. Zhang, M. E. Kozelkova, L. Isaacs, R. Nishiyabu, Y. Kubo and P. Anzenbacher, Jr., J. Am. Chem. Soc., 2012, 134, 20021; (c) T. Minami, N. A. Esipenko, A. Akdeniz, B. Zhang, L. Isaacs and P. Anzenbacher Jr., J. Am. Chem. Soc., 2013, 135, 15238. 10 Review, see: (a) J. J. Lavigne and E. V. Anslyn, Angew. Chem., Int. Ed., 2001, 40, 3118; (b) P. Anzenbacher, Jr., P. Lubal, P. Bucek, M. A. Palacios and M. E. Kozelkova, Chem. Soc. Rev., 2010, 39, 3954; current examples, see: (c) N. A. Esipenko, P. Koutnik, T. Minami, L. Mosca, V. M. Lynch, G. V. Zyryanov and P. Anzenbacher, Jr., Chem. Sci., 2013, 4, 3617; (d) P. Anzenbacher, Jr., Y. Liu, M. A. Palacios, T. Minami, Z. Wang and R. Nishiyabu, Chem.–Eur. J., 2013, 19, 8497; (e) Y. Liu, T. Minami, R. Nishiyabu, Z. Wang and P. Anzenbacher, Jr., J. Am. Chem. Soc., 2013, 135, 7705. 11 A. A. Tripolskaya, E. A. Mainicheva, T. V. Mit’kina, O. A. Geras’ko, D. Yu. Naumov and V. P. Fedin, Russ. J. Coord. Chem., 2005, 31, 768. 12 A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Huxley, C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97, 1515. 13 (a) A. S. Tippens, S. V. Davis, J. R. Hayes, E. C. Bryda, T. L. Green and C. A. Gruetter, Inflammation Res., 2004, 53, 390; (b) M. A. Perez-Amador, J. Leon, P. J. Green and J. Carbonell, Plant Physiol., 2002, 130, 1454. 14 K. L. Goa, M. Haria and M. I. Wilde, Drugs, 1997, 53, 1081. 15 D. L. Massart, B. G. M. Vandeginste, L. M. C. Buydens, S. De Jong, P. J. Lewi and J. Smeyers-Verbeke, Handbook of Chemometrics and Qualimetrics, Elsevier, Amesterdam, 1997.

Chem. Commun., 2014, 50, 61--63 | 63

"Turn-on" fluorescent sensor array for basic amino acids in water.

Amino acids and their derivatives are recognized and analyzed in water using a turn-on fluorescent cucurbituril based sensor array. Multivariate analy...
2MB Sizes 0 Downloads 0 Views