Langmuir 2000, 16, 7801-7804
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Influence of the Spacer Length of Glycolipid Receptors in Polydiacetylene Vesicles on the Colorimetric Detection of Escherichia coli Zhanfang Ma, Jinru Li, and Long Jiang* Laboratory of Colloid Interface Science, Center for Molecular Science, Chinese Academy of Sciences, De Wai Bei Sha Tan, Da Tun Road 3A, Beijing 100101, P. R. China, and The State Key Laboratory of Transducer Technology, Institute of Electronics, Chinese Academy of Sciences, Beijing 100080, P. R. China
Jie Cao Department of Microbiology, Beijing Medical University, Beijing 100083, P. R. China
P. Boullanger Laboratoire Chimique Organique 2, Universit Lyon 1, C. P. E. Lyon, France Received January 28, 2000. In Final Form: July 11, 2000 In the present study, the influences of the spacer length of the glycolipid receptors as well as the different diacetylenic lipid matrixes in color changeable polydiacetylenic vesicles on the detection of Escherichia coli (E. coli) were studied. The experimental results demonstrated that the glycolipids with longer spacer would be benefit to detecting E. coli. As a matrix lipid for colorchangeable polymer vesicles to detect E. coli, 2,4-heneicosadiynoic acid (HCDA), which has a shorter hydrophobic chain than 2,4-tricosadiynoic acid (TCDA), was more sensitive. The color change of the polymer vesicle solution using HCDA or TCDA as matrix was rapid, changing about 70-90% within 20 s, and nearly finishing in 2 min.
Introduction
Chart 1
It has been reported1-4 that polydiacetylenic vesicles functionalized with glycolipids are effective colorimetric biosensors. Conjugated polymers are just beginning to be extended to biological applications, especially in the case that the chromic transitions are utilized for the detection of biological macromolecules. We have previously reported that polymer vesicles using 2,4-tricosadiynoic acid (TCDA) as the matrix lipid and dioctadecyl glyceryl ether-β-glucoside as the receptor could make a biosensor to detect Escherichia coli (E. coli).3 In the present paper, we report the influence of the spacer length of glycolipid molecules and different diacetylenic lipids on the detection of E. coli. The chemical structures of glycolipids, TCDA and 2,4heneicosadiynoic acid (HCDA), are shown in Chart 1. Experimental Section TCDA and HCDA were purchased from Dojindo Laboratories (Kumamoto, Japan) and Tokyo Chemical Industry Co. Ltd. (Tokyo Japan), respectively. They were used without further purification. 3,6-Ethaoxa-4-cholest-2-acetamido-2-desoxy-β-D-glucopyranoside (DL1), 3,6,9,12-tetraoxa-10-cholest-2-acetamido-2-desoxyβ-D-glucopyranoside (DL3), and 3,6,9,12,15-pentaoxa-13-cholest2-acetamido-2-desoxy-β-D-glucopyranoside (DL4) were synthesized in Lyon by Boullanger. * Corresponding author. Tel.: 86-10-64888171. Fax: 86-1064879375. E-mail:
[email protected]. (1) Charych, D. H.; Cheng, Q.; Reichert, A.; Kuziemko, G.; Stroh, M.; Nagy, J. O.; Spevak, W.; Stevens, R. C. Chem. Biol. 1996, 3, 113. (2) Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585. (3) Ma, Z. F.; Li, J. R.; Liu, M. H.; Cao, J.; Zou, Z. Y.; Tu, J.; Jiang L. J. Am. Chem. Soc. 1998, 120, 12678. (4) Cheng, Q.; Stevens, R. C. Adv. Mater. 1997, 9, 481.
All surface pressure (π)-area (A) isotherms were obtained by FACE surface pressure meter HBM (made in Japan) at 20 ( 0.5 °C. The pressure sensor has a resolution of 0.1 mN/m. Glycolipids used were dissolved in a mixture of chloroform and methanol (2:1 in molar ratio). For each isotherm experiment, 100 µL of a 1.0 mM sample was spread on water subphase waiting 10 min for solvent evaporation before compression. Similarly, the same amount of sample was spread on E. coli aqueous solution (2 × 106/mL), waiting 1 h for full adsorption of E. coli before compression. The barrier was compressed at a speed of 20 cm2/ min. The procedures of the mixed vesicle preparation and the colorimetric assay were proceeded according to the method described elsewhere3. A 1 × 108/mL E. coli suspension was used to test.
10.1021/la000126+ CCC: $19.00 © 2000 American Chemical Society Published on Web 09/08/2000
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Figure 1. Isotherms of DLn monolayers on the water subphase with (dashed line) and without (solid line) E. coli: A, DL1; B, DL3; C, DL4.
Results and Discussion Unpolymerized vesicles composed of HCDA or TCDA and DLn were prepared in dark room. Then the system was polymerized under UV light irradiation at 254 nm. DLn molecules without unsaturated bond in their hydrophobic chain do not participate in the polymerization, whereas it was easy to polymerize for TCDA or HCDA under the same conditions and to form polydiacetylenic structure. As shown in our previous work,3,5 glycolipids could be inserted into colorchangeable polydiacetylenic vesicles based on noncovalent binding. When the amount of glycolipids was more than 0.1 (glycolipids to total lipids in molar ratio), it was difficult to form well-polymerized vesicles. Figure 1 shows the π-A curves of DLn monolayers with different spacer lengths. It is obvious that at high surface pressure all the analogues have the same critical surface area, ca. 40 Å2, similar to that of cholesterol itself.6 This indicates that at high surface pressure all spacers sink into the subphase bulk and make no contributions to the surface area, while at low surface pressure the number of spacer did have an effect on the surface area. The longer the spacer, the larger the surface area. This suggests that a longer spacer molecule could occupy a larger molecular area before compression. Figure 1 also tells us that the combination of E. coli with sugar headgroup did not increase the surface area much, indicating the reaction was taken place in the subphase bulk. According to the results of the π-A curves and the results obtained previously,5 a schematic diagram of the (5) Ma, Z. F.; Li, J. R.; Jiang, L. Langmuir 1999, 15, 489. (6) MRS International Meeting on Advanced Materials; Materials Research Society: New York, 1989; Vol. 1.
Figure 2. Schematic diagram of a polydiacetylenic vesicle.
polymerized vesicles composed of DLn/HCDA can be supposed as shown in Figure 2. All polydiacetylenic vesicles composed of DLn/HCDA and DL3/TCDA appeared blue, which arises from the eneyne conjugated system that comprises the polymer backbone of the polydiacetylene matrix.7 Diacetylene polymerization occurs only when the material is in a highly ordered state.8,9 The polymeric backbone formed is composed of alternative double and triple bonds, and the extent of conjugation depends on its conformation.10,11 If the effective conjugation length is reduced due to strain and torsion imposed onto the backbone, its color turned from blue to red.12-17 The backbone distortion can be induced by order-disorder transitions in the side chains through (7) Wegner, G. J. Polym. Sci. Part B 1979, 9, 133. (8) Polydiacetylenes; Cantow, H.-J., Ed.; Springer-Verlag: Berlin, 1994. (9) Shostakovskii, M. F.; Bogdanova, A. V. The Chemistry of Diacetylenes; John Wiley & Sons: New York, 1974. (10) Dobrosavljevic, V.; Stratt, R. M. Phys. Rev. B 1987, 35, 2781. (11) Eckhardt, H.; Boudreaux, D. S.; Chance, R. R. J. Chem. Phys. 1986, 85, 4116. (12) Deckert, A. A.; Fallon, L.; Kiernan, L.; Cashin, C.; Perrone, A.; Encalarde, T. Langmuir 1994, 10, 1948. (13) Kuriyama, K.; Kikuchi, H.; Kajiyama, T. Langmuir 1996, 12, 2283. (14) Berman, A.; Ahn, D. J.; Lio, A.; Salmeron, M.; Reichert, A.; Charych, D. H. Science 1995, 269, 515. (15) Sheth, S. R.; Leckband, D. E. Langmuir 1997, 13, 5652.
Colorimetric Detection of Escherichia coli
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Figure 3. Visible absorption spectra of polydiacetylenic vesicle solution before (solid line) and after (dashed line) addition to E. coli.
Figure 5. Dependence of the colorimetric response of DLn/ HCDA (a), and DL3/HCDA and DL3/TCDA (b) vesicle solutions on detecting time.
Figure 4. Colorimetric response curve of DLn/HCDA (a), DL3/ HCDA and DL3/TCDA (b) vesicles upon incubation with E. coli.
temperature,18,19 mechanical stress,20-23 pH,24 and solvent.25,26 In our case, the mechanism of color change for (16) Lio, A.; Reichert, A.; Ahn, D. J.; Nagy, J. O.; Salmeron, M.; Charych, D. H. Langmuir 1997, 13, 6524. (17) Lio, A.; Reichert, A.; Ahn, D. J.; Nagy, J. O.; Salmeron, M.; Charych, D. H. J. Vac. Sci. Technol. B 1996, 14, 1481. (18) Chance, R. R.; Patel, G. N.; Witt, J. D. J. Chem. Phys. 1979, 71, 206. (19) Shibata, M.; Kaneko, F.; Aketagawa, M.; Kobayashi, S. Thin Solid Films 1989, 179, 433. (20) Nallicheri, R. A.; Rubner, M. F. Macromolecules 1991, 24, 517. (21) Tashiro, K.; Nishimura, H.; Kubayashi, M. Macromolecules 1996, 29, 8188. (22) Tomioka, Y.; Tanaka, N.; Imazeki, S. J. Chem. Phys. 1989, 91, 5694.
polydiacetylenic structure is due to perturbation, i.e., mechanical stress.27 The suspension rapidly turned from blue to red after exposure to E. coli (dispersed in 0.85% aqueous solution of sodium chloride) as shown in Figure 3. No color change was observed when 0.85% aqueous solution of sodium chloride alone was added to the same system. Similarly, vesicles without DLn did not change color within several minutes after exposure to E. coli. Figure 4 shows the influence of the chain length of glycolipds on the colorimetric response (CR)28 of polydiacetylene vesicles. The measurement time was 2 min for each curve. It has been found that for DLn/HCDA polymer vesicles the sensitivity order for detecting E. coli is DL4/ HCDA > DL3/HCDA > DL1/HCDA as shown in Figure 4a. This fact proved the validity of the mechanical perturbation assumption. It is well-known that the ethenoxy group in spacer [-(CH2CH2O)n-] is hydrophilic, which makes a spacer extending into the water solution and forms a hard rod. According to the leverage principle, (23) Mitra, V. K.; Risen, W. M. J. Chem. Phys. 1977, 66, 2731. (24) Mino, N.; Tamura, H.; Ogawa, K. Langmuir 1992, 8, 594. (25) Chance, R. R. Macromolecules 1980, 13, 396. (26) Chu, B.; Xu, R. Acc. Chem. Res. 1991, 24, 384. (27) Lim, K.; Heeger, A. J. J. Chem. Phys. 1985, 82, 522.
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DL4 with longer spacer has a longer arm of force, which exerts a larger force on the polydiacetylene structure when they combine with E. coli. For the HCDA matrix, the visible spectrum of the polymer vesicle solution showed the absorption maximum at about 635 nm before exposure to E. coli and 520 nm after exposure to E. coli, respectively. For the TCDA matrix, however, the absorption maximum at about 667 nm before exposure to E. coli and 572 nm after exposure to E. coli can be measured. These results showed that both polydiacetylene vesicles were sensitive to E. coli. For HCDA and TCDA as matrix, the sensitivity order for detecting E. coli is DL3/HCDA > DL3/TCDA as shown in Figure 4b. This suggested that as matrix lipid for (28) Colorimetric response (CR). To quantity the response of a vesicle solution to a given amount of E. coli, the visible absorption spectrum of the vesicle solution without E. coli was analyzed as follows: B0,520 ) I0,520/I0,635; B0,635 ) I0,635I0,520, where B0,520 and B0,635 are defined as the intensity of absorption at 520 nm divided by the adsorption of 635 nm, and as the intensity of absorption at 635 nm divided by the adsorption of 520 nm, respectively. The vesicle solution which was exposed to E. coli was analyzed in the same way as follows: Bv,520 ) Iv,520/Iv,635; Bv,635 ) Iv,635/Iv,520, where Bv,520 and Bv,635 represent the new ratio of absorbance intensities after incubation with E. coli. The colorimetric response (CR) of a vesicle solution is defined as follows: ∆B520 ) Bv,520 - B0,520; ∆B635 ) B0,635 - Bv,635, CR ) ∆B635 + ∆B520. Similarly, for TCDA as matrix lipid CR ) ∆B667 + ∆B552.
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polymer vesicles, HCDA was more sensitive than TCDA. It is beyond our expectation because these two monomers have almost the same structure except TCDA has 18 CH2 and HCDA has 16 CH2. The color change of the polydiacetylenic vesicle solution was about 70-90% within 20 s, and nearly finished in 2 min as shown in Figure 5. This result showed that for polymer vesicles using TCDA or HCDA as matrix lipids the detecting speed was very rapid. Conclusion As receptors for polymer vesicles to detect E. coli, glycolipids with longer spacer were more sensitive in detecting E. coli, i.e., DL4/HCDA > DL3/HCDA > DL1/ HCDA. Polymer vesicles formed by the HCDA matrix, which has a shorter hydrophobic chain than TCDA, was more sensitive in the detection of E. coli. This work proved that the vesicles using HCDA or TCDA as matrix could change their color when biological molecules adsorbed on the receptors locating at the interface of these vesicles. Acknowledgment. This work was financed by grants from the Chinese Academy of Sciences (KL 951-A1-501-05). LA000126+
