Lithocholic acid – DMXAA chemical

Liquid crystals-based sensor for the detection of lithocholic acid coupled with competitive host-guest inclusion

Hui Maa, Qi Kangb, Tao Wangc, Jianhong Xiaoc, Li Yua,⁎

Keywords:
Liquid crystals
Sodium dodecyl sulphate Lithocholic acid
β-Cyclodextrin
Host-guest interaction

A B S T R A C T

A simple, low-cost and label-free strategy for detecting lithocholic acid (LCA) was designed at the liquid crystals (LCs)/aqueous interface via competitive host-guest inclusion. In this method, sodium dodecyl sulfate (SDS) was initially adsorbed on the ﬂuid interface and induced LCs to adopt the homeotropic ordering. Inclusion com- plexation of SDS and β-cyclodextrin (β-CD) disturbed interaction between LCs and SDS and evoked LCs to keep a tilted alignment. When injecting LCA into the miXed solution of SDS and β-CD, SDS excluded from the cavity of β-CD by competitive host-guest inclusion and could be re-adsorbed at the LCs/aqueous interface, resulting in the orientational transition of LCs from tilted to homeotropic state. Correspondingly, a bright-to-dark optical re- sponse was observed under polarized optical microscope (POM). The as-prepared LCs-based sensor could detect LCA as low as about 2 μM in aqueous solution. Moreover, the practicability of the approach was validated by monitoring the known amount of LCA in human urine. This work oﬀers an appealing approach for the detection of LCA which has a great potentiality in clinical diagnosis.

1. Introduction

Lithocholic acid (LCA), a secondary bile acid generating from the 7α-dehydroXylation of chenodeoXycholic acid, is known to be a toXic endobiotics [1,2]. Elevated level of LCA makes a signiﬁcant contribu- tion to carcinogenesis process [3,4]. Colon cancer is attributed to the accumulation of LCA reabsorbed poorly into enterohepatic circulation in the colon [5–7]. LCA concentration of 5 to 10 μM is also found in patients who suﬀer from chronic cholestatic liver disease [8]. There- fore, the establishment of LCA assay method provides useful clinical information for the diagnosis of relevant disease. However, just a few analytical methods were employed to achieve the determination of LCA, such as chromatography-mass spectrometry colorimetry [8,9] and ﬂuorometry [10]. Although these existing methods can identify LCA with high sensitivity, they encounter some drawbacks, e.g. costly in- strumentations, complex operation procedures and electric power, etc. Therefore, there is still a challenge to develop a novel, convenient and cost-eﬀective strategy for determination of LCA. β-CD is a cylinder-shaped host molecule with seven glucose subunits connected by α-(1, 4) glycosidic linkages [11,12]. The hydrophobic cavity of β-CD endows it with special capacity to form host-guest in- clusion complexes with a variety of suitably-sized substrates in aqueous medium. As reported previously, LCA and β-CD could form inclusion complex [10,13], which exhibited much stronger aﬃnity to the binding sites than other complexes formed by surfactants (e.g. SDS, DTAB) and β-CD. Hence, we attempted to construct an analytical platform for LCA detection on account of a competitive host-guest complexation.

LCs were considered as responsive materials. Some pioneering work of Abbot’s group has successfully demonstrated the LCs are very sen- sitive to the presence of surfactants [14,15]. The adsorption of proteins [16,17], lipids [18,19], DNA [20–22] and polymers [23,24] at the LCs/ aqueous interface also trigger the orientational transition of LCs, which are concomitantly transduced into alteration in the measurable optical signals. Brake et al. were the ﬁrst to propose the use of LCs for reporting biological binding events [25]. Recent studies have reported that LCs can also respond to fundamental interactions between antibody-antigen [26–28], surfactant-protein [29,30] and host-guest [31–33], etc. Herein a convenient, inexpensive and label-free LCs-based sensor was developed to detect LCA associated with competitive host-guest inclusion interactions. The adsorption of SDS at the LCs/aqueous in- terface was prevented due to its inclusion into β-CD. LCA could pre- dominate SDS to enter into the cavity of β-CD. As a result, the extruded SDS from the cavity of β-CD was laden at the interface and induced LCs to undergo a tilted-to-homeotropic ordering transition, corresponding to a bright to dark shift in the optical images. Based on this mechanism, the detection limit of LCA is ∼2 μM. The LCs sensor could also be used to identify LCA in human urine. This work opens a potential route for detection of LCA in practical application. Nematic liquid crystal, 4- cyano-4-pentylbi-phenyl (5CB), was utilized as LCs material. The che- mical structures of main compounds employed are shown in Fig. S1.

2. Experimental

2.1. Materials

Octyltrichlorosilane (OTS, 95%), 5CB (99%), D-(+)-glucose (ACS Grade), L-ascorbic acid (99%), potassium carbonate (K2CO3, ≥99.5%), urea (99%) and uric acid (98%) were purchased from J&K Scientiﬁc Co., Ltd. SDS (99%) was provided by Alfa Aesar (China) chemicals Co.,Ltd. β-CD (96%) was obtained from Aladdin Chemistry Co., Ltd. of China. Sodium hydroXide (NaOH, ≥96.0%) and sodium chloride (NaCl, ≥99.5%) were provided by Sinopharm Chemical Reagent Co., Ltd. LCA (> 98.0%) was produced by Tokyo Chemical Industry Co., Ltd. was dissolved in aqueous solutions at ∼pH 8 (adjusted with NaOH aqueous solution). Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O, ≥99.0%) and disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O, ≥99.0%) were obtained from Tianjin Damao Chemical Reagent Company of China. Copper grids were bought from Zhongjingkeyi Technology Co., Ltd of China. All the chemicals were used without further puriﬁcation. Ultrapure water (18.25 MΩ cm) was obtained with the Ulupure system and used in all the experiments.

2.2. Preparation of glass microscope slides

According to previous literature [34–36], glass microscope slides were cleaned in “piranha solution” (70% H2SO4/30% H2O2) for 30 min
at 80 ℃ (Warning: do not store the lotion in closed containers; should be operated with extreme caution). After sequential rinsing with water, ethanol, and methanol, the slides were dried under inert atmosphere with N2 and stoved overnight at 110 ℃. Then clean glass slides were soaked in the solution of heptane containing OTS for 30 min, followed by rinsing the slides using methylene chloride and drying under N2.

2.3. Fabrication of LC cells

Copper specimen grids were placed onto the OTS-coated glass substrates. Initially 5CB was heated to isotropic phase (> 35 ℃) and then dispensed onto the grid. Capillary tube (20 μL) was employed to remove the excess LC to form a uniform thin ﬁlm. Next, the 50 μL sample was introduced into the optical cell at ∼25 ℃. Each assay was operated at least three times.

2.4. Optical observation of LCs appearances

The optical images of these samples were captured with POM (XPF- 800C, Tianxing, Shanghai, China). Each image was obtained by a 2.5×
objective lens and a digital camera (TK-9301EC, JVC, Japan) at room temperature. The bright area coverage ratio (Br) of the optical ap- pearance was obtained by employing the Adobe Photoshop CS 6 via calculating the piXel percentage in the bright area to the total LC region. Br of the complete bright or dark was designated as 1 or 0, respectively.

2.5. Preparation of SDS/β-CD complex
SDS/β-CD complex was prepared according to the method pre- viously reported [32]. Brieﬂy, SDS aqueous solution with a ﬁXed con- centration was added to diﬀerent concentrations of β-CD aqueous so- lutions with equal volume at room temperature. The miXture was continuously stirred for 5 h at 45 ℃ and then cooled to room tem- perature and sonicated for 12 h.

2.6. Source and treatment method of urine

Urine samples were carefully taken from healthy individuals over a 24 h period and stored at 4 ℃. Since there is no or inﬁnitesimal LCA existing in the urine of healthy subjects [37], known amounts of LCA were added to the urine samples to simulate the urine of patients who suﬀer from cholestasis.

2.7. Surface tension

Surface tension measurements were performed on model Sigma 700 force tensiometer (Biolin Scientiﬁc, Finland) to evaluate the surface activities of samples. The Wilhelmy plate method was used to de- termine the surface tension data. After every measurement, the Wilhelmy plate was completely washed and heated in the ﬂame to re- move the residues of solutions. The surface tension values were de- termined at least ﬁve times and the mean value was considered. The thermostatic water bath was employed to control the experimental temperature.

2.8. Isothermal titration calorimetry

Isothermal titration calorimetry (ITC) was conducted on a Microcal VP-ITC apparatus with reference power of 10 μcal/s. 29 Injections of β- CD samples were added dropwise to the SDS or LCA solution. The miXture was stirred with 307 rpm at 25 ℃.

3. Results and discussion

3.1. Construction of LCs sensor based on SDS/β-CD complexes
Eﬀect of β-CD or SDS on the alignment of LCs at the ﬂuid interface was ﬁrstly investigated. Bright optical images of 5CB were presented when contacting with β-CD aqueous solutions in Fig. S2. There was no pronounced change observed in the optical signal with the increment of β-CD concentration, which implies that β-CD itself fails to inﬂuence the orientation of 5CB. As for SDS, bright appearance was still exhibited in the presence of lower SDS concentration (Fig. S3a–c). The orientation of LCs could turn to completely dark appearance when the concentration of SDS reached 0.25 mM or higher (Fig. S3d–f), indicative of home- otropic alignment of 5CB. These results were similar to that reported in previous work [14,31]. In consideration of the forthcoming experiment, we must ensure that the SDS concentration was not below 0.25 mM in the miXture when induced isopyknic LCA solution, because there could be no bright-to-dark transition captured under POM even though all of SDS was excluded from the cavity of β-CD. Therefore, 0.50 mM SDS after miXing with β-CD aqueous solution was selected as the optimum concentration to establish the LCs-based sensor. To evaluate the concentration of β-CD for construction of LCs-based sensor in the presence of 0.50 mM SDS, LCs textures under POM were ﬁrstly observed when 1.00 mM SDS solution was miXed with the same volume, diﬀerent concentrations of β-CD aqueous solutions (0.50, 1.00, 1.60, 2.00, 3.00 and 4.00 mM). As can be seen from Fig. 1a, black op- tical image appeared in the presence of 0.25 mM β-CD. Fig. 1b–f shows that β-CD at concentration of 0.50 mM or higher in the miXture could cause the optical images of LCs to achieve the dark-to-bright shift, associated with a homeotropic-to-tilted state alteration in the alignment of LCs at the ﬂuid interface.

In previous reports [38–40], surface tension measurements were also used to infer molecular interactions of host-guest complex. In our work, we employed surface tension technique to further ascertain the concentration of β-CD required to completely include 0.50 mM SDS in the cavities (viz. almost without free SDS). The surface tension value of 0.50 mM SDS solution was determined to be 60.12 mN m−1. In the case of β-CD aqueous solutions, the surface tension data display no change with the increasing concentration (Fig. 2, red bars), almost close to the surface tension of water. The surface tension values of SDS+β-CD miXed solutions (CSDS = 0.50 mM; Cβ-CD = 0.25, 0.50, 0.80, 1.00, 1.50, 2.00 mM) show a continuous increase at ﬁrst and then reach a plateau region when the concentration of β-CD ≥ 1.00 mM (Fig. 2, blue bars). Appearance of constant surface tension values suggest that host-guest inclusion of SDS into β-CD has reached saturation when existed 1.0 mM β-CD in the SDS and β-CD miXture. That is to say, almost all of SDS were into the cavities of β-CD and no free SDS existed in solution. Combined with the results of POM, it follows that host-guest inclusion com- plexation between SDS and β-CD is responsible for the orientational transition of LCs at the ﬂuid interface. Furthermore, the miXed solution (0.50 mM SDS, 1.00 mM β-CD) was conﬁrmed to establish the LCs- based sensor.

3.2. Detection of LCA in aqueous solution

POM images of LCs show a bright coloring when immersed in dif- ferent concentrations of LCA aqueous solutions without SDS and β-CD
miXture (Fig. S4), which suggests that LCA itself could not inﬂuence the tilted alignment of 5CB. Afterwards, in the presence of SDS and β-CD miXture, after transferring various concentrations of LCA solutions onto the LC interface the optical signal responses were depicted in Fig. 3. Furthermore, Br of correlative LCs images was calculated by Adobe Photoshop CS 6 (Fig. 4). Obviously, bright optical images of LCs were still maintained in contact with below 2 μM LCA (Fig. 3a, b). Yet a tiny change of optical appearance was captured at CLCA =2 μM (Fig. 3c). Dark patches appeared at CLCA = 20 μM (Fig. 3d), and the black region grew bigger at CLCA = 100 μM (Fig. 3e). In the case of CLCA = 200 μM, the optical image became uniformly black within 2 h (Fig. 3f). Hence based on the above results, the LCs-based sensor could detect LCA as low as about 2 μM. In addition, we inferred that occurrence of tilted-to- homeotropic state transition for LCs alignment was due to the sub- stituent LCA for SDS. The excluded SDS molecules from the cavities of β-CD may be adsorbed again on the ﬂuid interface, which induces 5CB to adopt the homeotropic ordering.

3.3. Detection of LCA in human urine samples

It is reported that patients suﬀering from cholestasis contain ele- vated levels of LCA [41]. Therefore, the content of LCA in human urine can provide signiﬁcant clinical information for the diagnosis of cho- lestasis. Interfering species such as inorganic salt (NaCl, K2CO3, NaH2PO4 and Na2HPO4), glucose, ascorbic acid, urea and uric acid present in human urine may aﬀect the detection of LCA in the sensor. Whereupon POM images of LCs incubated with SDS and β-CD miXture in the presence of 1 mM interference aqueous solutions were observed and shown in Fig. 5. Only bright optical images were observed in these aqueous solutions (Fig. 5a–h). Therefore, the LCs-based sensor enables
to monitor LCA in human urine without interference from other com- ponents in such environment. Furthermore, the sensor was applied to detect LCA in human urine samples. Various amounts of LCA were added to the urine samples. Fig. 6 shows the optical images of LCs individually observed in the absence and presence of LCA and the corresponding histogram for Br of LCs images associated with the concentrations of LCA was given in Fig. 7. Apparently, LCs displayed bright optical appearance after transferring healthy person’s urine onto the interface which incubated with the miXture of SDS and β-CD (Fig. 6a). When diﬀerent con- centrations of LCA were then introduced into the sensor, the achieved experimental results (Fig. 6b–f) were similar to those in aqueous solu- tions (Fig. 3). Detection limit of the LCA was determined to be around 2 μM in human urine and closed to the lowest detection concentration of LCA aqueous solutions. These observations suggest that the LCs- based sensor could provide an access to monitor LCA in human urine, which is fascinating for the real applications.

3.4. Detection mechanisms of LCA

In view of the experimental results, we speculated that competitive host-guest complexation between host (β-CD) and guest (SDS or LCA) molecules played a crucial role in the LCs-based sensor. In order to further explore the interaction mechanism, we employed ITC to quan- titatively characterize the host-guest interaction between β-CD and SDS or LCA, respectively. The recorded titration data are shown in Fig. 8Aa and Ba for β-CD/SDS and β-CD/LCA complexes, respectively. The host- guest combination between β-CD and SDS or LCA was exothermic process and exothermic heat of the latter was much stronger than that of the former. As the injections continued, the binding of β-CD with the corresponding guest molecules gradually became saturated and the exothermic heat gradually decreased. When the β-CD was saturated with SDS or LCA, no binding occurred and only heat of dilution was observed in Fig. 8Aa and Ba. Moreover, Fig. 8Ab and Bb were sepa- rately derived from the area integral of each peak of Fig. 8Aa and Ba, which were ﬁtted by computer simulation using the “one set of sites”
model [42]. In sharp contrast, a smooth arc-shaped isotherm for β-CD/ SDS complexation and a typical S-shape curve with an abrupt transition for β-CD/LCA host-guest pair were severally obtained. The individual association ratio (N) of β-CD to SDS or LCA was calculated to be 0.998 and 1.32, both of which is close to 1.0. The corresponding binding constant (Ka) is 1.15 × 104 M−1 and 6.28 × 105 M−1, respectively, which is separately in agreement with the association constants reported previously [43,44]. Therefore, it can be deduced that the strength of host-guest interaction between β-CD and LCA is much stronger than that of β-CD with SDS. So LCA is capable of expelling SDS from the cavity of β-CD due to the competitive inclusion complexation, which is consistent with our conjecture. Based on this, a schematic representation of LCA detection me- chanism is depicted in Fig. 9. SDS as the guest molecule was initially included into β-CD, which prevented it from being adsorbed at the LCs/ aqueous interface and cause LCs to adopt the tilted ordering (Fig. 9a). While in the presence of LCA solution, LCA would compete against SDS and ﬁnally gain entry into the cavity of β-CD. The excluded SDS dec- orates the ﬂuid interface and induces LCs to undergo an ordering transition from tilted (Fig. 9a) to homeotropic (Fig. 9b) state, corre- sponding to the bright (Fig. 9c) to dark (Fig. 9d) shift in the optical images.

4. Conclusions

The LCs-based sensor for LCA detection was fabricated using a copper grid cell with SDS and β-CD miXture, originating from the competitive host-guest inclusion. The optimum concentration for SDS and β-CD to construct the sensor was 0.50 mM and 1.00 mM, respec- tively, which was ascertained by POM and surface tension techniques. The detection limit of LCA was around 2 μM whether in aqueous so- lution or in human urine. The possible detection mechanism of LCA was due to the dominant inclusion of LCA/β-CD, the expelled SDS from the cavity of β-CD was again adsorbed at the LCs/aqueous interface and subsequently caused the ordering transition of 5CB from tilted to homeotropic state. Correspondingly, the bright-to-black alteration in the optical appearance of LCs was observed under POM. This work provides a convenient, low-cost and label-free assay for the detection of LCA which shows potential and promising prospects in sensing appli- cations.

Acknowledgments
This work was supportedby Natural Science Foundation of China (No. 21373128), Scientiﬁc and Technological Projects of Shandong Province of China (No. 2014GSF117001).

Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2018.09.071.

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