Article pubs.acs.org/ac
Polydiacetylene Liposomal Aequorin Bioluminescent Device for Detection of Hydrophobic Compounds Ryoko Yamamoto, Shigehiko Takegami,* Atsuko Konishi, Hikari Horikawa, Sayumi Yonezawa, and Tatsuya Kitade Department of Analytical Chemistry, Kyoto Pharmaceutical University, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan S Supporting Information *
ABSTRACT: In this study, a polydiacetylene liposomal aequorin bioluminescent device (PLABD) that functioned through control of the membrane transport of Ca2+ ions was developed for detecting hydrophobic compounds. In the PLABD, aequorin was encapsulated in an internal water phase and a calcium ionophore (CI) was contained in a hydrophobic region. Membrane transport of Ca2+ ions across the CI was suppressed by polymerization between diacetylene molecules. On addition of an analyte, the membrane transport of Ca2+ ions across the CI increased, and Ca2+ ions from the external water phase could diffuse into the internal water phase via the CI, which resulted in bioluminescence of the aequorin. Lidocaine, procaine, and procainamide were used as model compounds to test the validity of the detection mechanism of the PLABD. When each analyte was added to a suspension of the PLABD, bioluminescence from the aequorin in the PLABD was observed, and the level of this bioluminescence increased with increasing analyte concentration. There was a linear relationship between the logarithm of the analyte concentration and the bioluminescence for all analytes as follows: R = 0.89 from 10 nmol L−1 to 10 mmol L−1 for lidocaine, R = 0.66 from 10 nmol L−1 to 100 μmol L−1 for procaine, and R = 0.74 from 100 nmol L−1 to 100 μmol L−1 for procainamide. Compared to the traditional colorimetric method using polydiacetylene liposome, the PLABD was superior for both the sensitivity and dynamic range. Thus, PLABD is a valid, simple, and sensitive signal generator for detection of hydrophobic compounds that interact with PLABD membranes.
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direct colorimetric detection of the influenza virus using PDALS functionalized with sialic acid.13 Since then, biosensors using PDALS have been developed to detect many analytes, including cholera toxin,14 bacterial pore-forming toxin,15 phospholipases,16 nucleic acids,17 lipopolysaccharide,18 ionophores,19 organophosphates,20 phosphinothricin acetyltransferase,21 and cyclodextrins.22 A recent report described a PDALS array system combining a colorimetric method with a machinelearning statistical algorithm for disease diagnosis using plasma samples.23 However, most of the developed PDALS colorimetric/fluorometric detection methods have some limitations, such as low sensitivity and narrow dynamic range. This is because colorimetric, spectrometric, and fluorometric signal generation from PDALS results from a large conformational change in the PDA membrane. In this study, a polydiacetylene liposomal aequorin (AQ) bioluminescent device (PLABD) was developed for detection of hydrophobic compounds that interact with PLABD membranes. The proposed detection mechanism of the PLABD is shown in Figure 1. First, the diacetylene liposome
ecause of its unique electrical and optical properties, polydiacetylene (PDA) has been widely investigated for application in sensing materials. PDA is a polymer that has a conjugated ene−yne bond between adjacent diacetylene molecules, which is formed by photopolymerization using UV irradiation. Because of the presence of the π-conjugated polymer chain, PDA can absorb visible light and usually appears blue in color1,2 and emits red fluorescence.3,4 Furthermore, PDA shows an obvious chromatic transition from blue to red in response to external stimuli, such as changes in the pH5 or temperature,6 mechanical stress,7 solvents,8 and interactions with biological analytes.9 This phenomenon is thought to occur because of shortening of its conjugation length following conformational change in the PDA. However, the mechanism for this has not been clarified yet. Among the available sensing materials, PDA micelles and PDA liposome (PDALS), which forms by self-assembly of amphiphilic diacetylene monomers through photopolymerization, have attracted increasing interest in several research fields. PDA micelles have been investigated for biomedical applications, including tumor imaging and drug delivery.10−12 Meanwhile, PDALS is a well-known signal generator for biosensors used in colorimetric/fluorometric detection of chemical and biological analytes. Charysh et al. first reported © XXXX American Chemical Society
Received: November 27, 2015 Accepted: May 5, 2016
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DOI: 10.1021/acs.analchem.5b04500 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 1. Detection mechanism of PLABD for analytes.
Figure 2. Chemical structures of analytes, lidocaine (LCN), procaine (PCN), and procainamide (PCA), used in this study.
were dissolved in an appropriate volume of chloroform in a round-bottom flask. The CM was used as the Ca2+ ionophore. After the removal of the chloroform under a stream of nitrogen gas, a thin layer of the mixture was left on the bottom of the flask. Diethyl ether (6 mL) was added, and the flask was placed in the ultrasonic bath (SONOREX DIGITEC DT-255H, BANDELIN electronic GmbH & Co. KG, Berlin, Germany). Then, 1.8 mL of a 10 mmol L−1 aqueous solution of (NH4)2SO4 containing 5% sucrose, which was pH adjusted to 11 using 1 mol L−1 NaOH, was added to the flask. After this, 200 μL of a 200 μg mL−1 aqueous solution of AQ (JNC Corp., Yokohama, Japan) was carefully added to the sodium sulfate aqueous phase. The mixture was sonicated for 30 s in the ultrasonic bath to form a water-in-oil emulsion. After the removal of diethyl ether under reduced pressure, 10 mL of the 10 mmol L−1 aqueous solution of (NH4)2SO4 containing 5% sucrose was added to the residue. The suspension was sonicated for 2 min in the ultrasonic bath. Residual diethyl ether in the suspension was completely removed under reduced pressure to leave DALS. The DALS suspension was transferred to a quartz cell. A suspension of PLABD was obtained by irradiating the cell with UV light (λ < 254 nm, 0.43 mW cm−2) for a set time. The final concentrations of PCDA, CM, and AQ were 1.0 mmol L−1, 1.67 μmol L−1 and 0.17 μmol L−1, respectively. DALS and PDALS without CM and AQ were also prepared by the same method. Effect of Different UV Irradiation Periods. PLABD suspensions were prepared with different UV irradiation periods (5, 10, and 15 min). The DALS and PLABD suspensions were diluted with the 10 mmol L−1 aqueous solution of (NH4)2SO4 containing 5% sucrose (pH 11) to have a PCDA concentration of 250 μmol L−1. Measurements of Particle Size and ζ Potential of DALS and PLABD. The DALS and PLABD suspensions were diluted with the 10 mmol L−1 aqueous solution of (NH4)2SO4 containing 5% sucrose (pH 11) with a 1:1 (v/v) dilution for the particle size measurements and a 1:1000 (v/v) dilution for the ζ potential measurements. The mean particle sizes and their distributions for DALS and PLABD were determined by
(DALS) was formed by self-assembly of diacetylene monomers. The DALS encapsulated AQ, which is a photoprotein with a high emission quantum yield, in an internal water phase, and a calcium ionophore (CI) in a hydrophobic region. The PLABD was prepared by photopolymerization of DALS under UV irradiation. We hypothesize that the PLABD will function as follows. In the PLABD, membrane transport of Ca2+ ions across the CI will be suppressed by the packed planar PDA membrane, which means that any Ca2+ ions present in the external water phase will not be transported into the internal water phase via the CI. In this case, bioluminescence will not be observed from the AQ. However, when an analyte is added, it will interact with the PDA membrane to cause a conformational change. After this change, Ca2+ ions will be transported from the external water phase into the internal water phase via the CI according to Ca2+ ion concentration gradient. This will result in AQ bioluminescence on binding of Ca2+ ions to AQ. This bioluminescence confirms the presence of the analyte. The main goal of this study was to demonstrate the validity of the detection mechanism of the PLABD for hydrophobic compounds. Therefore, PLABD was applied to the detection of lidocaine (LCN), procaine (PCN), and procainamide (PCA) as model analytes (Figure 2). The PLABD response and sensitivity for these analytes were compared to those of a traditional colorimetric method using PDALS. As a first approach, a simple PLABD was prepared with a single polymerized lipid so that the results could be easily interpreted. Also, because there have been many reports of PDALS preparation from 10,12-pentacosadiynoic acid (PCDA) in the literature, the behavior of this PDALS is well-understood. Thus, we chose to use PCDA to prepare the PLABD in this study.
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EXPERIMENTAL SECTION Preparation of the PLABD. PLABD was prepared using the reverse-phase-evaporation method.24 First, 4.5 mg of PCDA (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and 4.0 μL of a dimethyl sulfoxide stock solution of 5 mmol L−1 calcimycin A23187 (CM; Sigma−Aldrich Chemical, St. Louis, MO, USA) B
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moieties of adjacent PCDA molecules under UV irradiation, color changes of DALS and PLABD suspensions after UV irradiation for 5, 10, and 15 min were examined (Figure 3).
dynamic light scattering using a Nicomp 380 analyzer (Particle Sizing Systems Inc., Santa Barbara, CA, USA). The reported particle sizes and their distributions are volume-weighted. ζ potentials were measured using a Zeecom ZC-3000 analyzer (Microtec Co., Ltd., Chiba, Japan) based on electrophoresis. Measurement of Absorption Spectra. Absorption spectra were measured for the PDALS prepared by UV irradiation for 10 min. Aliquots (500 μL) of LCN aqueous solutions with various concentrations (0.1−100 mmol L−1) were added to 500 μL of the PDALS suspension in 2 mL volumetric flasks. After mixing for 1 min, a sufficient volume of the 10 mmol L−1 aqueous solution of (NH4)2SO4 containing 5% sucrose (pH 11) was added to reach a PCDA concentration of 250 μmol L−1. The sample solutions were transferred to cuvettes with a 1 cm path length. The absorption spectra of the sample solutions were measured against the 10 mmol L−1 aqueous solution of (NH4)2SO4 containing 5% sucrose (pH 11) as a reference solution. Spectra were recorded using a U3310 spectrophotometer (Hitachi, Ltd., Tokyo, Japan) with a spectral range of 500−700 nm, bandpass of 2 nm, wavelength interval of 0.1 nm, and scan speed of 60 nm min−1. Measurement of AQ Bioluminescence in DALS and PLABD. The PLABD prepared by UV irradiation for 10 min was used in this experiment. First, 150 μL of the DALS or PLABD suspension was transferred to a test tube and then diluted with 150 μL of the 10 mmol L−1 aqueous solution of (NH4)2SO4 containing 5% sucrose (pH 11) to a PCDA concentration of 500 μmol L−1. After mixing for 30 s, the test tube was placed in the sample holder of a luminometer (Lumat LB9507, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Following the injection of 100 μL of a 20 μmol L−1 aqueous solution of CaCl2 into the test tube, the bioluminescence of AQ was measured for 15 s every 0.5 s. Bioluminescent intensity (BI) values were obtained by integrating each 15 s bioluminescent signal. Measurement of AQ Bioluminescence from PLABD with Model Analytes. The PLABD prepared by UV irradiation for 10 min was used in this experiment. First, 150 μL of the PLABD suspension was transferred to a test tube. To eliminate bioluminescence of AQ in the external water phase of PLABD, 30 μL of a 200 μmol L−1 aqueous solution of CaCl2 was added to the test tube. Then, the mixture was diluted with 120 μL of the 10 mmol L−1 aqueous solution of (NH4)2SO4 containing 5% sucrose (pH 11) to PCDA and CaCl 2 concentrations of 500 and 20 μmol L−1, respectively. After mixing for 30 s, the test tube was placed in the sample holder of the luminometer. After another 30 s, 100 μL of an aqueous solution of the analyte with an appropriate concentration was injected into the test tube. The BI of AQ was measured as described above. Since the amount of AQ encapsulated in the internal water phase of PLABD was different for each PLABD preparation, the BI values were normalized by conversion to BI ratios (BIRs) as follows: BIR =
Figure 3. Color changes of (a) DALS and PLABDs with different irradiation periods of (b) 5, (c) 10, and (d) 15 min.
DALS was a white suspension (Figure 3a), while the PLABD suspensions after irradiation for 5 and 10 min were blue (Figure 3b,c). Therefore, the diacetylene moieties of adjacent PCDA molecules polymerized under UV irradiation and the ene−yne conjugate bond formed to give PLABD. However, after 15 min of UV irradiation, the PLABD suspension was reddish-purple in color (Figure 3d). This indicates that a conformational change of the polymeric structure of PLABD occurred when the UV irradiation was extended from 10 to 15 min. Thus, 10 min of UV irradiation was used for preparation of PLABD. Characterization of DALS and PLABD. The particle sizes and their distributions, and ζ potentials of DALS and PLABD are shown in Table 1. The particle size of DALS showed a Table 1. Particle Size and Size Distribution, and ζ Potential Values of DALS and PLABDa particle size ± size distribution (μm) DALS PLABD a
peak 1
peak 2
ζ potential (mV)
0.71 ± 0.10 0.66 ± 0.10
12.90 ± 1.86 12.73 ± 2.71
−27.6 ± 8.2 −23.3 ± 6.7
The values represent the mean ± SD (n = 3).
bimodal distribution around 0.71 and 12.90 μm with a volumeweighted distribution ratio of 2:98 (see Supporting Information Figure S-1). The power of the ultrasonic bath was not high enough to obtain nanoscale DALS. The DALS had a negative ζ potential of about −28 mV because the carboxylic (COO−) groups of the PCDA molecules formed DALS. The particle sizes and their distributions, and ζ potentials of DALS and PLABD (i.e., before and after UV irradiation) were similar. In a previous report, the particle size of a PDA vesicle was smaller than that of diacetylene monomers because of polymerization and packing.13 By comparison, in the present study, the UV polymerization had no effect on the measured physicochemical parameters. Color Change of PDALS with Model Analytes. To examine the response of PDALS to model analytes, color changes of PDALS were observed in the presence of LCN (Figure 4a). The color of the PDALS suspensions clearly changed from light blue to light red with increasing LCN concentration. Absorption spectra (Figure 4b) of the PDALS suspensions were also measured in the presence of LCN at the same concentrations as in Figure 4a. As the LCN concentration increased, the absorbance of PDALS at 645 nm decreased and that at 550 nm increased. The results indicate that LCN at concentrations > 0.25 mmol L−1 influences the conjugate length and conformation of the conjugated ene−yne bond between adjacent PCDA molecules, and this induces the
BI at an appropriate analyte concentration mean BI at analyte concentration of 10 μmol L−1
A calibration curve for each analyte was prepared by plotting the BIRs against the logarithms of the analyte concentrations.
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RESULTS AND DISCUSSION Effect of UV Irradiation Period. To confirm if DALS was converted to PLABD by polymerization of the diacetylene C
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aqueous solution. However, there was a large difference between the curves for DALS and PLABD, with the maximum bioluminescent signal being smaller for PLABD than for DALS. This result indicates that the membrane transport of Ca2+ ions across the CI is suppressed by polymerization of the PCDA membrane. This result can be explained as follows. AQ exists in both the external and internal water phases of DALS and PLABD. Because membrane transport of Ca2+ ions across the CI is not suppressed in DALS, the CI can bind with Ca2+ ions near the membrane surface of DALS, and these Ca2+ ions then diffuse into the PCDA membrane and are released to the internal water phase. Therefore, in the DALS suspension, both the external and internal water phases can emit bioluminescence. By contrast, in the PLABD suspension, Ca2+ ions cannot be transported to the internal water phase via the CI because the membrane transport of Ca2+ ions across the CI is suppressed by the polymerization. Therefore, only AQ in the external water phase emits bioluminescence. These results show that the polymerization of the PCDA membrane can suppress the membrane transport of Ca2+ ions across the CI in PLABD. Kinetics and Calibration of AQ Bioluminescence in PLABD with the Model Analytes. Figure 6 shows changes in Figure 4. (a) Color change and (b) absorption spectra of PDALS suspensions in the presence of various LCN concentrations: (1) 0, (2) 0.025, (3) 0.25, (4) 2.5, (5) 6.25, (6) 12.5 and (7) 25 mmol L−1.
change in color of the PDALS suspension from light blue to light red. By contrast, when PCN and PCA were added to the PDALS suspensions at concentrations similar to that of LCN, slight color changes or spectral changes were observed (data not shown). The traditional colorimetric method using ultraviolet−visible spectroscopy has insufficient sensitivity to detect these drugs. Comparison of AQ Bioluminescence in DALS and PLABD. To confirm whether the membrane transport of Ca2+ ions across the CI was suppressed by the polymerization of PCDA molecules in PLABD, changes in the bioluminescent signal of AQ with time were measured for DALS and PLABD after the addition of a 20 μmol L−1 aqueous solution of CaCl2. Their changes are shown in Figure 5. Strong and short bioluminescence from AQ was observed in both the DALS and PLABD suspensions just after the addition of the CaCl2 Figure 6. Changes in the bioluminescent signal of AQ in PLABD suspensions at various times after the addition of LCN aqueous solutions of different concentrations.
the bioluminescent signal of AQ with time for PLABD after the addition of LCN aqueous solutions with various concentrations (1 nmol L−1 to 10 mmol L−1). A short bioluminescent signal from the AQ in PLABD was observed with each LCN solution just after its addition. The level of this bioluminescent signal increased with increasing LCN concentration. The same results were also obtained for PCN and PCA. These results show that the external Ca2+ ions were transported into the internal water phase via the CI in PLABD after interaction of PLABD with the analyte. This allowed the internal AQ to emit bioluminescence because of its binding with the Ca2+ ions transported by the CI. These results suggest that our hypothesis of how the detection mechanism of PLABD works is reasonable and that PLABD can generate a signal in response to a slight conformational change of the conjugated ene−yne bond from interaction with analytes. This signal generation will occur even at relatively low
Figure 5. Changes in the bioluminescent signal of AQ with time for DALS and PLABD suspensions after the addition of 20 μmol L−1 aqueous solution of CaCl2. D
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be considered as follows. According to an earlier study,25 binding of the analyte to the surface of PLABD and/or permeation of the analyte through the membrane will cause a conformational change in the conjugated ene−yne bond in PLABD. The pKa values of LCN, PCN, and PCA are 7.9, 8.9, and 9.2, respectively.26,27 Usually, the percentage of the neutral form, A, and protonated form, AH+, of an ionizable drug in a sample solution can be calculated from the Henderson− Hasselbalch equation (i.e., log([A]/[AH+]) = pH − pKa). Under the present experimental conditions at pH 11, the proportions of the neutral forms for LCN, PCN, and PCA were approximately 100%, 99%, and 98%, respectively. Therefore, all of the analytes studied will mostly exist as neutral forms in the sample solutions. Meanwhile, the membrane surface of PLABD is negatively charged at pH 11 as shown in Table 1. The protonated form of an analyte could bind electrostatically to the negatively charged membrane surface of PLABD, but as detailed above, under the present experimental conditions the analytes will be neutral not protonated. Therefore, binding of protonated analytes with the surface of PLABD will not contribute to the conformational change of the conjugated ene−yne bond. However, the logarithms of the n-octanol/water partition coefficients (log P) of the analytes used in this study are 2.26 for LCN, 0.88 for PCN, and 1.92 for PCA.28 The neutral form can penetrate into the hydrophobic region of PLABD much easier than the protonated form, and this will cause direct conformational change of the conjugated ene−yne bond formed between adjacent PCDA molecules in PLABD. Because LCN is more hydrophobic than PCN and PCA, LCN more effectively induced this conformational change than PCN or PCA. To elucidate whether the neutral form contributes to the conformational change, further study with LCN was performed at pH 9. Under the same LCN concentration in Figure 4a, no color change of PDALS suspensions was observed at pH 9 (see Figure S-2). This result demonstrates that LCN caused more significant conformational change of PDALS at pH 11 than at pH 9. The proportion of the neutral form of LCN at pH 9 is approximately 93% and is lower than that at pH 11. Therefore, it is indicated that the conformational changes were induced by the neutral form of LCN which can penetrate near the conjugated ene−yne bond between adjacent PCDA molecules in the hydrophobic region of PDALS and affect the conjugate length. In addition, the PLABD showed better linearity, higher sensitivity, and a larger dynamic range at pH 11 than at pH 9 (R = 0.77 and linear range of 100 nmol L−1 to 10 mmol L−1; see Figure S-3). These results show that as the proportion of the neutral form in the sample solution increased (i.e., from pH 9 to 11), a better response was obtained with the PLABD. This supports our above proposal that the penetration of the neutral form of an analyte into the hydrophobic region of PLABD plays an important role in inducing conformational change of the conjugated ene−yne bond. Thus, these results show that the PLABD can generate a signal for detection of hydrophobic compounds that interact with PLABD membranes.
concentrations that would not show a visible color change in other methods. The calibration curves for LCN, PCN, and PCA are shown in Figure 7. The calibration curves for the three analytes showed a
Figure 7. Calibration curves of (a) LCN, (b) PCN, and (c) PCA with PLABD. Error bars indicate the standard deviations of three replicate experiments.
linear relationship between the logarithm of the analyte concentration and the BIR values as follows: R = 0.89 and linear range of 10 nmol L−1 to 10 mmol L−1 for LCN, R = 0.66 and linear range of 10 nmol L−1 to 100 μmol L−1 for PCN, and R = 0.74 and linear range of 100 nmol L−1 to 100 μmol L−1 for PCA. From comparison of these results with the colorimetric method in Figure 4, the PLABD showed higher sensitivity (nanomolar level) and a larger dynamic range (at least three orders of magnitude greater) than the colorimetric method. Therefore, our PLABD method is more superior in both sensitivity and dynamic range for these analytes than the traditional colorimetric method. When comparing the results for the different analytes, the PLABD for LCN showed better linearity, higher sensitivity, and a larger dynamic range than for PCN and PCA. This result can
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CONCLUSIONS In conclusion, a PLABD was developed that could be used for detection of the model analytes LCN, PCN, and PCA. Compared with the traditional colorimetric method, which has low sensitivity and a narrow dynamic range (millimolar level), the PLABD showed higher sensitivity and a larger dynamic range (nanomolar to micromolar levels). Thus, the E
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(10) Gravel, E.; Ogier, J.; Arnauld, T.; Mackiewicz, N.; Ducongé, F.; Doris, E. Chem. - Eur. J. 2012, 18, 400−408. (11) Yang, D. J.; Zou, R.; Zhu, Y.; Liu, B.; Yao, D.; Jiang, J.; Wu, J.; Tian, H. Nanoscale 2014, 6, 14772−14783. (12) Neuberg, P.; Perino, A.; Morin-Picardat, E.; Anton, N.; Darwich, Z.; Weltin, D.; Mely, Y.; Klymchenko, A. S.; Remy, J.-S.; Wagner, A. Chem. Commun. 2015, 51, 11595−11598. (13) Reichert, A.; Nagy, J. O.; Spevak, W.; Charych, D. J. Am. Chem. Soc. 1995, 117, 829−830. (14) Pan, J. J.; Charych, D. Langmuir 1997, 13, 1365−1367. (15) Ma, G.; Cheng, Q. Langmuir 2005, 21, 6123−6126. (16) Jelinek, R.; Okada, S.; Norvez, S.; Charych, D. Chem. Biol. 1998, 5, 619−629. (17) Jung, Y. K.; Kim, T. W.; Kim, J.; Kim, J. M.; Park, H. G. Adv. Funct. Mater. 2008, 18, 701−708. (18) Wu, J.; Zawistowski, A.; Ehrmann, M.; Yi, T.; Schmuck, C. J. Am. Chem. Soc. 2011, 133, 9720−9723. (19) Kolusheva, S.; Shahal, T.; Jelinek, R. J. Am. Chem. Soc. 2000, 122, 776−780. (20) Lee, J.; Seo, S.; Kim, J. Adv. Funct. Mater. 2012, 22, 1632−1638. (21) Jung, S.-H.; Jang, H.; Lim, M.-C.; Kim, J.-H.; Shin, K.-S.; Kim, S. M.; Kim, H.-Y.; Kim, Y.-R.; Jeon, T.-J. Anal. Chem. 2015, 87, 2072− 2078. (22) Brockgreitens, J.; Ahmed, S.; Abbas, A. J. Inclusion Phenom. Macrocyclic Chem. 2015, 81, 423−427. (23) Kolusheva, S.; Yossef, R.; Kugel, A.; Katz, M.; Volinsky, R.; Welt, M.; Hadad, U.; Drory, V.; Kliger, M.; Rubin, E.; Porgador, A.; Jelinek, R. Anal. Chem. 2012, 84, 5925−5931. (24) Szoka, F., Jr.; Papahadjopoulos, D. Proc. Natl. Acad. Sci. U. S. A. 1978, 75, 4194−4198. (25) Shin, M. J.; Kim, Y. J.; Kim, J.-D. Soft Matter 2015, 11, 5037− 5043. (26) Volpe, P.; Palade, P.; Costello, B.; Mitchell, R. D.; Fleischer, S. J. Biol. Chem. 1983, 258, 12434−12442. (27) Saenger-van de Griend, C. E.; Ek, A. G.; Widahl-Naesman, M. E.; Andersson, E. K. M. J. Pharm. Biomed. Anal. 2006, 41, 77−83. (28) Hansch, C.; Leo, A.; Hoekman, D. Exploring QSAR, Vol. 2; American Chemical Society: Washington, DC, USA, 1995.
PLABD could be used to easily and sensitively detect hydrophobic compounds. Because the current PLABD does suffer from low selectivity and specificity, it may need some separation procedures such as the extraction of analytes in the practical assay for real samples. Therefore, our future challenges will focus on development of the selective and specific assay without any separation procedures. For hydrophobic compounds, we have examined a PLABD array system which includes several PLABDs prepared using different polymerized lipids. When real sample containing an analyte is added to the PLABD array system on a microplate, the signal emission pattern obtained from the PLABD array system should be specific to each analyte because the affinity of an analyte to this range of polymerized lipids will be unique. Meanwhile, in the case of hydrophilic compounds which can interact on the surface of PLABD membrane, PLABD will be able to enhance both selectivity and specificity through modification of its surface with biological recognition units that would facilitate strong and specific binding to target molecules. These PLABD systems are currently undergoing testing, and the results will be reported in the future.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04500. Particle size distributions of DALS and PLABD, color change of PDALS with LCN at pH 9, and calibration curve of LCN with PLABD at pH 9 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +81 75 595 4659. Fax: +81 75 595 4760. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partly supported by grants from Leave a Nest Co., Ltd. with Microtec. Co., Ltd. and Takeda Science Foundation and by a subsidy from the Ministry of Education, Culture, Sports, Science and TechnologySupported Program for the Strategic Research Foundation at Private Universities, 2013−2018 (Grant No. S1311035).
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