Simple and Effective Label-Free Capillary Electrophoretic Analysis of

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Simple and Effective Label-Free Capillary Electrophoretic Analysis of Sugars by Complexation Using Quinoline Boronic Acids Takuya Kubo, Koichi Kanemori, Risa Kusumoto, Takayuki Kawai, Kenji Sueyoshi, Toyohiro Naito, and Koji Otsuka Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00998 • Publication Date (Web): 24 Apr 2015 Downloaded from http://pubs.acs.org on April 28, 2015

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Analytical Chemistry

Simple and Effective Label-Free Capillary Electrophoretic Analysis of Sugars by Complexation Using Quinoline Boronic Acids

Takuya Kubo,*,† Koichi Kanemori,† Risa Kusumoto,† Takayuki Kawai,‡ Kenji Sueyoshi,§ Toyohiro Naito,† Koji Otsuka†

†

Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

‡

Laboratory for Integrated Biodevice Unit, Quantitative Biology Center, RIKEN, Chuo-ku, Kobe, Hyogo

650-0047, Japan §

Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University,

Sakai, Osaka, 599-8531, Japan

E-mail: [email protected]

ACS Paragon Plus Environment

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Abstract: An effective separation and detection for sugars by capillary electrophoresis (CE) using a complexation between quinolineboronic acid (QBA) and multiple hydroxyl structure of sugar alcohol is reported. We investigated the variation of fluorescence spectra of a variety of QBAs with sorbitol at wide range of pH conditions, and then found that 5-isoQBA strongly enhanced the fluorescence intensity by the complexation at basic pH condition. The other sugar alcohols having multiple hydroxyls also revealed the enhancement of the fluorescence intensity with 5-isoQBA, whereas the alternation of the intensity was not found in the sugars such as glucose. After optimization of 5-isoQBA concentration and pH of the buffered solution in CE analysis, 6 sugar alcohols were successfully separated in the order based on the formation constants with 5-isoQBA, which were calculated from the variation of the fluorescence intensity with each sugar alcohol and 5-isoQBA. Furthermore, the limit of detection for sorbitol and xylitol by the CE method were estimated at 15 µM and 27 µM, respectively.


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Introduction The quantitative analysis of sugars is important in clinical science and some of sugars in biosamples are utilized as bio-markers of the affections. For example, sialic acid is known as a marker of cancer cells, which is situated at the termination of the sugar chain on the surface of the cell, and one of carbohydrate antigen CA19-9 having sialic acid in the blood is usually checked as a marker of pancreatic cancer.1,2 Also, it is well known that the concentration of glucose in the blood is very important for the examination of diabetes mellitus3 and the structures of carbohydrates in antibody preparations have important roles for physiological activity and/or stability.4 Therefore, simple and effective detection method for sugars at high accuracy and sensitivity is commonly required. For the separation and detection of sugars, liquid chromatography (LC) with anion-exchange columns and ligand-exchange columns using the complex of metal ions have been used.5,6 However, LC analyses need a large amount of the samples and/or solvents for mobile phase, and time consumption, so that capillary electrophoresis (CE) is expected as an alternative method to overcome these drawbacks in LC. Although CE provides a small sample consumption, a high efficiency, and a short analysis time, most of sugars are electrically neutral and have no UV absorbance and/or fluorescent. Therefore, commonly used CE is not applicable for the analysis of sugars and certain derivatization is required. On the other hand, boronic acid easily generates a reversible organic complex with a cis-diol structure 7,8

and Czarnik et al. reported a fluorescent sensor for the detection of sugars.9 As further reports, a few

boronic compounds have been reported for higher selective and sensitive detection of sugars,10-14 and the separations by LC combined with boronic compounds have been also reported.15,16 According to the specific complexation property of boronic acids, a variety of studies including a drug delivery system,17 a catalysis,18 and stationary phase of CE or LC19,20 have been reported. Especially, the sensing of sugars by the changing of spectroscopic characterization depend on the complexation between a boronic acid and a sugar has attract attention.21-26 Recently, an alternative CE method using allylboronic acids has been developed.27,28 In this method, sugars were effectively separated and detected with 3-nitrophenylboronic acid due to the difference of ACS Paragon Plus Environment

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the electrophoretic mobility and UV-absorption, respectively. As the other related works, Wang et al. found the functionality of quinolineboronic acids (QBAs) including 5-QBA and 8-QBA for the detection of sugars by significant fluorescence changing due to the complexation.29,30 According to the specific fluorescent property of QBAs, a physicochemical property of QBAs and several applications regarding sensor technologies for sugars have been also reported in recent years.31-35 However, the fundamental studies regarding the complexation of QBAs and sugars have not been reported, and its applications to the CE analysis have not been completed. In the present study, we aim to develop an effective, selective and simple analysis method for the detection of sugar alcohols using CE. The optimizations of the conditions including pH and excitation/emission among a number of QBAs were carried out for the effective detection of sugar alcohols. Furthermore, we revealed the simple and effective CE separation/detection of sugar alcohols depending on the difference of the formation constants, which were estimated by the variations of the fluorescent intensity using 5-isoQBA and each sugar alcohol.


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EXPERIMENTAL SECTION Fundamental measurements on the complexation of QBAs and sugar alcohols Fluorescence spectra of QBA standards (see Figure S1) diluted with a 15 mM phosphate buffer at 2 µM (4-isoquinolineboronic acid, 4-isoQBA), 5 µM (5-isoquinolineboronic acid, 5-isoQBA), and 20 µM (3Quinolineboronic acid and 8-quinolineboronic acid, 3-QBA and 8QBA) were measured by a 1 cmlength quartz cell. Optimum wavelengths for excitation and emission in the complexation of QBAs and sugar alcohols were obtained with QBA/15 mM phosphate buffer and 10 mM or 1.0 mM of sorbitol aqueous solutions. The pH dependence for the fluorescent intensity of the complexes were measured at pH 4.8 to 10.0. The excitation and emission wavelengths were employed as follows; (ex/em), 3-QBA, 315/385 nm; 8-QBA, 315/420 nm; 4-isoQBA, 330/375 nm; 5-isoQBA, 273/347 nm, respectively. The fluorescence spectra based on each sugar alcohol including meso-erythritol, L-(−)-arabitol, xylitol, mannitol, galactitol, sorbitol, meso-erythritol, glucose, N-acetylneuraminic acid, mannose, and fructose were measured with 5-isoQBA (5 µM/15 mM phosphate buffer, pH 8.3). To obtain the electrophoretic mobility, µep, of each complex, CE analyses were carried out with/without 10 mM sorbitol at pH 3.0 to 12.0 as a background solution (BGS) using 10 ppm thiourea or 20 µM 5-isoQBA as the solutes, a fused silica capillary (50 µm i.d., total/effective length, 40/30 cm), 15 kV as an applied voltage, and 0.5 psi-5 s for a sample injection.

CE separation and detection of sugar alcohols with 5-isoQBA Optimizations of CE conditions were carried out with a fused silica capillary (50 µm i.d., total/effective length, 86.5/46.2 cm), 5-isoQBA/27 mM phosphate buffer as a BGS, 15 kV as an applied voltage, and a hydrodynamic injection (15 cm, 10 s). The fluorescence intensity was measured at 273/347 nm (ex/em) for all the sugar alcohol samples. For the separation of the mixture sample was carried out with the same conditions using 3 mM 5-isoQBA/27 mM phosphate buffer (pH 8.3) as a BGS with (100 µM) sorbitol,


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galactitol, mannitol, (250 µM) galactonic acid, (300 µM) xylitol, L-(−)-arabitol, and (500 µM) mesoerythritol. (see Figure S1)

RESULATS AND DISCUSSION Optimization of excitation/emission and pH in complexation of QBAs As fundamental studies are needed for the sensitive detection of sugar alcohols by complexation with QBAs, the excitation/emission and pH conditions were optimized. Figure S2 shows the excitation spectra of 3-QBA, 8-QBA, 4-isoQBA, and 5-isoQBA with 1.0 mM or 10 mM sorbitol solutions. Here, excess amount of sorbitol was employed to obtain the complete complexation of QBAs. As results, excitation wavelengths were selected for 3-QBA, 8-QBA, 4-isoQBA, and 5-isoQBA as 315 nm, 315 nm, 330 nm, and 273 nm, respectively. Additionally, the emission wavelength was also optimized by the evaluation of the maximum absorption wavelength with sorbitol (Figure S3). Finally, the optimal emission wavelengths for 3-QBA, 8-QBA, 4-isoQBA, and 5-isoQBA were confirmed as 385 nm, 420 nm, 375 nm, and 347 nm, respectively. By using these excitation/emission conditions, the effect of pH during the complexation of QBAs and sorbitol was evaluated. Figure 1-(a) shows the variation of the fluorescence intensity with/without sorbitol. As shown in this figure, higher pH condition is necessary for the complexation because the variations were not observed at lower pH. The results also indicated that as for detecting sugar alcohols 5-isoQBA and 4-isoQBA were superior to 3-QBA and 8QBA, since no intensity variation was observed in 3-QBA and 8QBA by sorbitol. Especially, 5-isoQBA provided a significant variation of the fluorescent intensity with sorbitol around pH 9.0. As shown in Figure 1-(b), the fluorescence spectrum of 5-isoQBA with 1.0 mM sorbitol at pH 9.0 was drastically changed compared to a free 5-isoQBA solution. According to these results, we concluded that 5-isoQBA was suitable for the sensitive detection of sugar alcohols in a basic condition.


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2E+08 20

emission intensity / 10−7 M−1

5 µM 5-isoQBA (ex, 273 nm; em, 347 nm) 2 µM 4-isoQBA (ex, 231 nm; em, 370 nm) 20 µM 3-QBA (ex, 315 nm; em, 385 nm) 20 µM 8-QBA (ex, 315 nm; em, 420 nm)

(a)

2E+08 15

1E+08 10

5E+07 5.0

0E+00 0

6

7

8

9

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700

(b) 600

with 1.0 mM sorbitol without sorbitol

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intensity

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400 300 200 100 0 300

350

400

450

500

wavelength / nm

Figure 1. Fluorescence intensity of QBAs toward pH and the variation of the spectra with sorbitol. (a) pH dependence of the fluorescence intensity of QBAs with sorbitol. Conditions, the phosphate buffers at pH 6 to 10 by detection (ex/em), 3-QBA, 315/385 nm; 8-QBA, 315/420 nm; 4-isoQBA, 330/375 nm; 5-isoQBA, 273/347 nm. Dotted lines show the result of QBAs itself without sorbitol. (b) the fluorescence spectra of 5-isoQBA at pH 9.0 with/without sorbitol of 1.0 mM.


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Fluorescence spectra of the complexes of sugar alcohols with 5-isoQBA The variations of the fluorescent spectra of the sugar alcohols with 5-isoQBA were measured. For 6 sugar alcohols, the variations of the intensity responsive to the number of hydroxyl groups and the configuration of vicinal hydroxyl groups were clearly observed as shown in Figure 2. As expected, boronic acid effectively recognized the orientation of vicinal hydroxyl groups like as cis-diol structures so that the highest intensity was obtained in sorbitol. Similarly, the other sugar alcohols allowed the variation of spectra by adding 5-isoQBA except glucose, which has no cis-diol structure. (see Figure S4(a)) Although the variation was not observed in meso-erythritol at 1.0 mM, a slight variation was confirmed when the concentration of meso-erythritol was increased at 10 mM. (see Figure S4-(b)) These results also corresponded that 5-isoQBA was suitable for the detection of sugar alcohols by the complexation with the vicinal hydroxyl groups structures in sugar alcohols. 600 with 1.0 mM sorbitol with 1.0 mM galactitol with 1.0 mM manitol with 1.0 mM xylitol with 1.0 mM arabitol with 1.0 mM meso-erythritol without carbohydrates

500

400

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300

200

100

0 300

350

400

450

wavelength / nm

Figure 2. Fluorescence spectra of the sugar alcohols with 5-isoQBA. The spectra were obtained with 5.0 µM 5-isoQBA /BGS (pH 8.3) by the excitation at 273 nm. ACS Paragon Plus Environment

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Estimation of pKa and the dissociation structures of 5-isoQBA The dissociation equilibriums shown in Figure 3-(a) and (b) can be identified independently, and the electrophoretic mobility, µep, is expressed as (eq-1) in CE analysis.

µep = µep1 x1 + (1-x2) µep2

(eq-1)

where µep1 and x1 are the electrophoretic mobility and mole fraction of a cationic QBA in equilibrium (a), x2 is the mole fraction of a neutral QBA, and (1- x2) and µep2 are mole fraction and the electrophoretic mobility of an anionic QBA in equilibrium (b), respectively. Furthermore, the dissociation constants, Ka1 and Ka2 are expressed as (eq-2) and (eq-3), respectively. (1 − x2 )[H + ] (eq-3) Ka2 = 　 x2

(1 − x1 )[H + ] (eq-2) K a1 = 　 x1 Then, µep can be expressed as (eq-4).

µ ep =

[H + ] Ka2 µ ep1 + µ ep2 + K a1 + [ H ] K a 2 + [H + ]

(eq-4)

In order to understand pKa and dissociation structure of 5-isoQBA and its complex, a CE analysis of 5isoQBA was carried out with/without sorbitol as a BGS at pH 3 to 12. Figure 4 shows the plots of the electrophoretic mobility against pH of the BGS. As a result, the electrophoretic mobilities of 5-isoQBA at acidic condition with or without sorbitol were slightly different in each other. We assumed that the complexation was not enough to form a negative charge, so that we concluded that the electrophoretic mobilities in acidic condition were too high for CE separation. The result of Figure 1-(a) also supported the assumption because the variation of the fluorescence intensity was not observed in lower pH conditions. On the other hand, the electrophoretic mobility of 5-isoQBA became negative at the neutral and basic conditions, and the value with sorbitol was lower than that of 5-isoQBA itself at pH 4 to 9. Especially, the difference in the electrophoretic mobility was larger in the region of pH 7 to 9. ACS Paragon Plus Environment

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Additionally, the association constants shown in Figure 3 were calculated by the results of Figure 4 and above equations. The each association constant including Ka1, Ka2, K’a1, and K’a2 was estimated at 4.9, 8.6, 4.2, and 6.8, respectively. According to these results, we expected that the effective CE analysis of sugar alcohols using 5-isoQBA under the condition of around pH 8.0 could be achieved.

Ka1

+ H3O+ (a)

+ H2O

+

Ka2

+

+ H3O+ (b)

2H2O R1

K’a1 ++

R1 O

+

O

R2 B

O

H2O

+ H3O+

(c)

+

(d)

N

R2 B

O

K’a2 N

+ 2H2O

H3O+

Figure 3. Prospective equilibrium states of 5-isoQBA with/without a sugar. Two-step dissociation of 5-isoQBA itself (a), (b), and with a sugar (c), (d). 3

2

µep / 10-4 cm2V-1s-1

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without sorbitol with 10 mM sorbitol

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−1

−2

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12

pH

Figure 4. Electrophoretic mobility of 5-isoQBA against pH. ACS Paragon Plus Environment

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Conditions: instrument, P/ACE MDQ; capillary, 50 µm i.d. × effective 30 cm; BGS, 15 mM phosphate buffer with/without 10 mM sorbitol; sample, 20 µM 5-isoQBA; injection, 5 psi-5 s; applied voltage, 15 kV; detection, ex/em (273/347 nm).

Optimization of 5-isoQBA concentration and pH in BGS for CE analysis of sugar alcohols As mentioned above, the suitable pH condition for the CE analysis of sorbitol using 5-isoQBA was neutral to basic. To obtain the optimum pH in the CE analysis for a number of sugar alcohols, a variety of sugar alcohols were analyzed under several pH conditions (pH 7.3 to 10.0). For the analyses, a 28 mM phosphate buffer containing 3 mM 5-isoQBA was used as a BGS. The concentration of each sugar alcohol was fixed at 500 µM and higher pH was adjusted by adding NaOH. According to the apparent electrophoretic mobility, µapp, of each sugar alcohol, the significant differences of µapp were not observed under higher pH conditions (pH > 9). On contrary, µapp of all the sugar alcohols were differed in each other at somewhat basic conditions (pH 7.5 to 9). (see Figure S5) It is assumed that most of sugar alcohols were formed the complex with 5-isoQBA under higher pH conditions, and then the negatively charged complexes provided similar electrophoretic mobilities. In consideration of these results and the variation of the fluorescence intensity (Figure 1), the suitable pH of 5-isoQBA containing BGS was fixed as 8.3. As well as the pH condition, the concentration of QBA is also important factor affecting the electrophoretic mobility. Therefore, the effect of the concentration of 5-isoQBA was also examined in the CE analysis using a variety of sugar alcohols. As a result of the comparison between µapp and the 5isoQBA concentration (1.0 to 4.0 mM), µapp of mannitol/xylitol and mannitol/galactitol were closed at 2.0 mM and 4.0 mM, respectively. Additionally, although the suitable differences of µapp were observed at 5-isoQBA of 1.0 mM, the fluorescence intensity was not enough for the sensitive detection of sugar alcohols in this concentration. (see Figure S6) Consequently, the concentration of 5-isoQBA in a BGS was fixed at 3.0 mM for the CE analyses.


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Estimation of the formation constants The formation constant between sugar and QBA can be described as below. Kf  → SQ S+Q ← 

Kf =

[SQ ] [S][Q]

S, sugar; Q, QBA

At the equilibrium state, the concentrations of the sugar and QBA are expressed as below.

[S] = [S]0 − [SQ ]

[Q] = [Q]0 − [SQ ]

where [S]0 and [Q]0 are the initial concentrations of the sugar and QBA, respectively. Additionally, the fluorescence intensity F is expressed as follows; F [Q]0 = F0 [Q] + F∞ [SQ] where F0 is the fluorescence intensity of QBA itself and F∞ is the maximum fluorescence intensity on the complexation with the sugar. Finnaly, F, F0, F∞, [S]0, [Q]0, and Kf are realized as the following equation (eq-5)

  F　− F0 F − F0 [Q ]0  = K f  [S]0 − F∞ − F F∞ − F0  

(eq-5)

To estimate Kf of each sugar alcohol with 5-isoQBA based on the equation, the fluorescence intensities of the sugar alcohols including meso-erythritol, L-(−)-arabitol, xylitol, mannitol, galactitol, and sorbitol with 5-isoQBA were measured by a fluorometer. Here, the fluorescence intensity of 5-isoQBA itself at 347 nm and the intensity with 1.0 mM 5-isoQBA were employed as F0 and F∞, respectively. Briefly, the fluorescence intensity with around 1.0 mM 5-isoQBA was stable, so that we considered that all the intensities were caused by the complete complexes of the sugar alcohol and 5-isoQBA. For example, the ACS Paragon Plus Environment

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variation of fluorescence spectra of sorbitol with 5-isoQBA is shown in Figure 5. Also, the fitting line based on eq-5 is shown in Figure 6. Accoding to these results, the formation constant, Kf, between sorbitol and 5-isoQBA was estimated as 6.8 × 102 M−1. Similarly, the fitting lines were prepared for the other sugar alcohols, and each Kf and the correlation coefficients are summarized in Table 1. 600

with 1.0 mM sorbitol (F∞)

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450

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Figure 5. The variation of fluorescence spectra of 5-isoQBA with sorbitol. The spectra were obtained with 5 µM 5-isoQBA/15 mM phosphate buffer (pH 8.3) using 16.7 µM, 33.3µM, 50 µM, and 1.0 mM sorbitol solutions.


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Figure 6. Approximate correlation line fitted to eq-5. Each fluorescence intensity was obtained from the same conditions as Figure 6.

Table 1. The estimated formation constants of the complexes between 5-isoQBA and sugar alcohols.

arabitol

xylitol

mannitol

galactitol

sorbitol

Kf (M )

mesoerythritol 3.5 ×102

1.1 ×103

2.8 ×103

3.6 ×103

4.4 ×103

6.8 ×103

R2

0.980

0.990

0.997

0.996

0.994

0.977

–1

All the Kf values and the coefficient of correlations were calculated from eq-5.

CE separation of sugar alcohols and limit of detection According to the optimized CE conditions, which were consisted of 3.0 mM 5-isoQBA/28 mM phosphate buffer (pH 8.3) as a BGS and the fluorescence detection at 273/347 nm (ex/em), the mixture of 6 sugar alcohols, catechol, and galactonic acid were analyzed. The electropherogram of the mixture sample is shown in Figure 7. As a result, all the solutes were successfully separated and detected without any derivatizations. Here, a negative peak of catechol was caused by the decrease of the fluorescence intensity based on the complexation. In brief, the intensity of the complex of catechol was lower than that of 5-isoQBA itself, so that the negative peak was detected.(see Figure S7) Interestingly, the stereoisomers including L-(−)-arabitol/xylitol and mannitol/galactitol/sorbitol were effectively separated by the method. Additionally, the migration order of these sugar alcohols corresponded to the estimated Kf values shown in Table 1. Therefore, we strongly suggest that the separation based on the strength of the complexation was accomplished in the developed CE method. Subsequently, the limit of detection (LOD) in the suggested CE method was estimated with sorbitol and xylitol at signal-to-noise ratio, S/N = 3. As results, the LOD for sorbitol and xylitol was estimated as 15 µM and 27 µM, respectively. Additionally, the linearity of the analytical curves for sorbitol and xylitol were also accepted even in the mixture samples as shown in Figure S8. From these results, the ACS Paragon Plus Environment

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suggested CE method can be used for the mixture sample without the inhibition of each complexation. The reason is considered that the concentration of 5-isoQBA was high enough compared to the concentration of sugar alcohols.

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5

1. meso-erythritol 2. arabitol 3. xylitol 4. Mannitol 5. galactitol 6. sorbitol 7. catechol 8. galactonic acid

6

2

8

0

–20

7 –40 12

14

16

18

20

22

24

Time / min Figure 7. CE separation of sugar alcohols with 5-isoQBA in BGS. CE conditions: capillary, fused silica capillary (50 µm i.d. × effective 46.2 cm); BGS, 3 mM 5isoQBA/28 mM phosphate buffer (pH 8.3); samples, (100 µM) 6. sorbitol, 4. mannitol, 5. galactitol, (200 µM) 7. catechol, (250 µM) 8. galactonic acid, (300 µM) 2. arabitol, 3. xylitol, (500 µM) 1. mesoerythritol / BGS, injection, 15 cm for 10 s; applied voltage, 10 kV; detection, em/ex (273 nm/347 nm)

CONCLUSION A simple and effective CE method for the detection of sugar alcohols baesd on the complexation of QBAs and the sugar alcohols was proposed. As results of the examination of a variety of QBAs for the effective complexation with sugar alcohols, 5-isoQBA allowed higher efficiency for the detection of sugar alcohols. After the optimization of the CE conditions with 5-isoQBA, the sugar alcohols were successfully separated and detected with the method. We also revealed that the migration order of sugar ACS Paragon Plus Environment

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alcohols in the CE method was corresponed to the formation contants of the complexes, which were estimated by the fundamental fluorescence measurements. Additionally, the LODs of sugar alcohols by this method were satisfied and superior to those obtained by the other previous studies. We believe that the proposed method in this study will be useful for the effective detection of multiple sugars in urine or blood samples.

ASSOCIATED CONTENT Supporting Information Structures of QBAs and sugars, optimization of the excitation and emission for QBAs with sorbitol, variation of the fluorescence spectra of sugars with 5-isoQBA, apparent electrophoretic mobility of sugars against pH, apparent electrophoretic mobility of sugars against 5-isoQBA concentrations, fluorescent spectrum of 5-isoQBA with catechol, analytical curves for sorbitol and xylitol by the optimized CE method. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT This research was partly supported by the Grant-in Aid for Scientific Research (No. 25620111 and 24350039) from the Japan Society for the Promotion of Science. We specially thank Prof. Philip BritzMcKibbin, Department of Chemistry and Chemical Biology, McMaster University for helpful discussion.


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Graphic Abstract


Simple and Effective Label-Free Capillary Electrophoretic Analysis of

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