Alignment of nematic liquid crystals decorated with gemini surfactants and interaction of proteins with gemini surfactants at fluid interfaces

Accepted Manuscript Alignment of Nematic Liquid Crystals Decorated with Gemini Surfactants and Interaction of Proteins with Gemini Surfactants at Fluid Interfaces Tongtong Tian, Qi Kang, Tao Wang, Jianhong Xiao, Li Yu PII: DOI: Reference:

S0021-9797(18)30175-9 https://doi.org/10.1016/j.jcis.2018.02.027 YJCIS 23299

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

23 November 2017 7 February 2018 8 February 2018

Please cite this article as: T. Tian, Q. Kang, T. Wang, J. Xiao, L. Yu, Alignment of Nematic Liquid Crystals Decorated with Gemini Surfactants and Interaction of Proteins with Gemini Surfactants at Fluid Interfaces, Journal of Colloid and Interface Science (2018), doi: https://doi.org/10.1016/j.jcis.2018.02.027

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Alignment of Nematic Liquid Crystals Decorated with Gemini Surfactants and Interaction of Proteins with Gemini Surfactants at Fluid Interfaces

Tongtong Tiana,b, Qi Kangc, Tao Wangd, Jianhong Xiaod and Li Yu a, b*

a

Key Laboratory of Colloid and Interface Chemistry, Shandong University, Ministry of Education, Jinan 250100, P.R. China

b

School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, PR China

c

College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, P. R. China

d

Petroleum Engineering Technology Research Institute of Shengli Oilfield, Sinopec, Dongying 257000, PR China.

Corresponding author: Prof. Dr. Li Yu Phone number: +86-531-88364807 Fax number: +86-531-88564750 E-mail address: [email protected]

Abstract A series of cationic gemini surfactants with diverse chemical structures, that is, imidazolium-based gemini surface active ionic liquids (gemini IM-SAILs) with different alkyl chain length or spacer length, viz. 1,s-bis(3-alkylimidazolium-1-yl) ethane bromide ([Cn-s-Cnim]Br2; s=2, n=6, 8, 10, 12, 16; n=12, s=2, 4, 6, 10), and quaternary ammonium-based gemini surfactants (gemini QASaa) with different symmetries, viz. 1,2-bisalkylquaternary ammonium bromide (m-2-n; m=12, 14, 16, n=8, 10, 12, m+n=24), were synthesized and utilized to decorate aqueous/liquid crystal interfaces (ALI). Initially, the optical response of the LCs changed from bright to dark after incubation with gemini IM-SAILs (except [C6-2-C6im]Br2) or gemini QASaa aqueous solutions, due to the formation of stable surfactant monolayers at the ALI. We verify that gemini IM-SAILs with shorter spacer or longer hydrophobic chains are more conducive to adsorption onto the interface, and gemini IM-SAILs form monolayers more easily than the corresponding monomers or gemini QASaa. Interestingly, a dark-to-bright shift in the optical image of the LCs subsequently occurred after the fluid interface decorated with the gemini surfactants came into contact with Bovine serum albumin (BSA), a negatively charged protein in neutral environments, whereas the optical appearance of LCs did not change upon addition of two other proteins with positive charge (viz. lysozyme and trypsin). Therefore, based on the different action mechanisms, a low-cost, label-free, and convenient LC-based sensing platform using the gemini surfactant-decorated LC interface was constructed for identification of the proteins with opposite charges. Keywords: Liquid crystal sensor; Imidazolium-based gemini surface active ionic liquid; Quaternary ammonium-based gemini surfactant; Protein; Opposite charge.

1. Introduction For decades, sensing platforms based on nematic liquid crystals (LCs), have drawn attention from far and wide [1]. The anchoring transition of LCs at the aqueous/LC interface is controlled by energy on the order of 10-2-10 -3 mJ·m-2, thus they can be outstandingly sensitive to external stimulus. Because of the convenient platform LC-based methods can provide, various chemical and biological events at fluid interfaces have been amplified and converted into optical signals that are visible to the naked eye under POM (polarized optical microscopy) [2-5]. Surfactants, as one kind of amphiphile, have been used extensively as decorators for the liquid-liquid interface of LC-based sensors [6-16]. For example, the alignments of LCs triggered by surfactants with various chemical structures were systematically exploited by Abbott’s group [6-9]. The hydrophobic effect between surfactant tail and LCs was found to play a significant part in the orientation of LCs at the ALI. Park et al. constructed a cholesterol biosensor upon adding β-cyclodextrin (β-CD) to the sodium dodecyl sulphate-decorated ALI [10]. Singh’s group developed a LC-based sensor for the

real

time

detection

of

mercuric

ions

(Hg2+)

using

potassium

N-methyl-N-dodecyldithiocarbamate [11]. Fang et al. designed LC droplets coated with β-CD-amphipathic tetradecyl trimethylammonium bromide compounds to detect cholic acid [12]. Our group previously built an ionic liquid/LC interface modified by 4-ethylazobenzene-4'-(oxy-hexyl) trimethyl ammonium bromide to carry out photo-control reversible and persistent alignment of LCs [13]. In early studies, although LC-sensing platforms in the presence of surfactants were widely reported, they were mostly limited to single-chained surfactants, with gemini surfactants seldom involved. Gemini surfactants possess two hydrophobic chains and two hydrophilic headgroups which are covalently linked through a spacer group [17]. Compared with conventional surfactants, their unique physico-chemical features, such as higher adsorption efficiency, lower critical micelle concentration and better wetting property, have put them in the spotlight [18]. To date, a variety of gemini surfactants [19-22]

have been designed and synthesized with diverse headgroups, various spacers and different aliphatic chain lengths. Bovine serum albumin (BSA) is a globular protein of great interest to researchers, with an isoelectric point (pI) of about 4.7. Thus, it has net negative charge in a water medium at neutral pH [19]. Many reports have proved that interactions of BSA with cationic gemini surfactants are stronger than those of conventional single-chained cationic surfactants. Electrostatic interaction and hydrophobic interaction are both considered as the major interaction forces between cationic surfactants and BSA [19-22]. Lysozyme, another protein receiving attention, is positively charged in neutral aqueous media due to its pI=11.4 [23]. The interactions between cationic surfactants and lysozyme were testified to take place mainly via hydrophobic interaction [23,24]. So far there are many reports on the interactions of surfactants with proteins in the bulk phase and at the air/water interface, yet the binding events occurring at other fluid interfaces have not been described. As is well known, proteins play a fundamental role in nutrition, immunity, enzyme and material transportation, etc [25-27]. Therefore, it is essential to develop various methods to identify or monitor them. This inspires us to investigate the orientation of LCs coupled to the interactions between proteins with different kinds of charges and cationic gemini surfactants at the ALI. In previous work, we investigated the absorption features of gemini surfactants at the ALI and found that gemini IM-SAIL ([C12-2-C12im]Br2) was much more readily adsorbed at the LC interface to form a self-assembled monolayer, compared to the corresponding

monomer

surfactant,

1-dodecyl-3-methylimidazolium

bromide

([C12mim]Br) [28]. In this continuation of our early work, we firstly studied the orientational transition of LCs at the ALI triggered by a series of cationic gemini surfactants with various chemical structures, namely, gemini IM-SAILs with different aliphatic or spacer length and gemini QASaa with different symmetries. Then imaging interactions at the ALI were successively explored between the negatively-charged protein (BSA), positively-charged proteins (lysozyme and trypsin) and multiple cationic surfactants (including gemini surfactants and their monomers). Ultimately proteins with different charge in neutral water were successfully distinguished with

the aid of LC-based platform and gemini surfactants. 2. Experimental Section 2.1 Materials Copper specimen grids (75 mesh, pitch = 340 µm, hole = 285 µm, bar = 55 µm,) were purchased from Beijing Gilder. Nematic liquid crystal 4-cyano-4'-pentylbiphenyl (5CB), N,N,N′,N′-tetramethylethylenediamine (99%), octadecyltrichlorosilane (OTS), heptane, dodecyltrimethyl ammonium bromide (DTAB, 99%), 1-bromohexane (99%), 1-bromooctane

(99%),

1-bromotetradecane

1-bromodecane

(99%),

(99%),

1-bromododecane

1-bromohexadecane(99%),

imidazole

(99%), (99%),

1,2-dibromoethane(99%), 1,4-dibromobutane(99%), 1,6-dibromohexane(99%) and 1,10-dibromodecane(99%) were obtained from J&K Scientific Co., Ltd., China. BSA was obtained from Shandong Aibo Technology Trade Co., Ltd., China. Lysozyme and trypsin were supplied by Shanghai Shifeng Biological Technology Co., Ltd., China. Isopropanol was obtained from Tianjin Kemiou Chemical Reagent Company of China. Acrylonitrile was purchased from Shandong Xiya Chemical Industry Co., Ltd. of China. Dichloromethane, sodium hydroxide, methanol, acrylonitrile, hydrogen peroxide (30% w/v), isopropyl alcohol and chloroform were all purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. Ultrapure water (κ=18.25 MΩ/cm, pH=6.5) was used to prepare aqueous solutions. 2.2 Synthesis of gemini IM-SAILs, gemini QASaa and [C12mim]Br The gemini IM-SAILs and gemini QASaa with different chemical structures [29-32] (Fig. 1) and [C12mim]Br [33] were synthesized according to the procedures reported previously. Their structures were ascertained by 1H NMR spectroscopy with a Bruker Advance 400 spectrometer. 1H NMR details and synthesis processes are presented in the Supporting Information. 2.3 Preparation of glass microscope substrates In this study, the preparation of glass microscope slides was in accordance with earlier reports [5]. First, “piranha solution” ( VH2SO4 : VH2O2 = 7 : 3 ) was used to clean the glass substrates for 30 min at 80 °C. Next, these slides were rinsed completely

with ultrapure water, ethanol, and methanol, then dried under a jet of N2 and heated at 110 °C overnight. The “piranha solution-cleaned” glass slides were then immerged in the OTS/heptanes solution for 30 min. Finally, they were bathed in methylene chloride and dried under a stream of N2. 2.4 Preparation of optical cells Copper specimen grids were cleansed sequentially in methylene chloride, ethanol, and methanol, dried under N2, and heated to 110 °C overnight. They were firstly placed onto the OTS-treated glass microscope substrates. Then, after dispensing about 1 µL of 5CB onto each copper specimen grid and removing the excess 5CB by a 20 µL capillary tube, 200 µL aqueous solutions of interest were introduced into optical cell. The schematic illustration of the experimental system is shown in Fig. S1 and all the results were repeated at least three times. 2.5 Optical examination of LC textures A polarized light microscope (Tianxing, XPF-800C, Shanghai, China) was applied to acquire the optical images of 5CB. All the images were obtained with a digital camera (JVC, TK-9301EC, Japan) and a 2.5 × objective lens.

Fig. 1 Chemical structures of symmetric gemini IM-SAILs and dissymmetric gemini QASaa.

3. Results and discussion 3.1 Effect of gemini surfactants with various structures on the orientation of 5CB.

Several gemini homologues with different alkyl chains, spacer, headgroups and symmetry (chemical

structures

shown

in

Fig.1),

viz.

gemini

IM-SAILs

([Cn-2-Cnim]Br2 , n = 6, 8, 10, 12, 16); [C12-s-C12im]Br2, s=2, 4, 6, 10) and gemini QASaa (m-2-n, m = 12, 14, 16; n = 8, 10, 12; m+n = 24), were synthesized and added to ALI, respectively, with an aim to investigate impact of gemini surfactants on the orientations of 5CB. 3.1.1 Effect of gemini surfactants with different alkyl chains length on the orientation of 5CB. We firstly determined if surfactant monolayers could be assembled at the ALI for all the gemini IM-SAILs with different aliphatic chain lengths. Fig. 2 presents the optical images of LCs when the fluid interface was incubated with different concentrations of [Cn-2-Cnim]Br2 (n = 6, 8, 10, 12, 16) aqueous solution. For [C6-2-C6im]Br2 within the concentration range (0-150 mM), the optical appearance of the LCs failed to change from bright to dark, indicative of planar birefringence of LCs at the ALI. Analogously, Abbott and co-workers observed parallel orientation of LCs within the studied concentration range for single-chained surfactants with short alkyl chain lengths, viz. octyltrimethylammonium bromide, N,N-dimethylferrocenyl alkylammonium bromides [8]. A possible reason is that the hydrophobic interaction between [C6-2-C6im]Br2 and LC molecules is too weak to change the anchoring of LCs.

Fig. 2 POM images of 5CB at the ALI in the presence of of [Cn-2-Cnim]Br2 ( n = 6, 8, 10, 12, 16) aqueous solutions with various concentrations.

However, spontaneous bright-to-dark transformation of LCs images was observed when adding [C8-2-C8im]Br2 (1 mM), [C10-2-C10im]Br2 (5 µM), [C12-2-C12im]Br2 (0.5 µM) and [C16-2-C16im]Br2 (0.1 µM) aqueous solutions to the ALI. The variation in the LCs optical appearance can be attributed to the formation of a self-assembled gemini IM-SAILs monolayer at the interface, which induces anchoring of LCs from planar to homeotropic orientation due to the hydrophobic interactions between the tail chains of gemini IM-SAILs and LCs [8]. Our results show clearly that the threshold concentration (Cthreshold), at which the bright optical appearance of the LCs converts to dark reduced as the number of carbon atoms in the gemini IM-SAILs increases from 8 to 16. Such concentration-dependence of optical response is in agreement with previous reports [8], and can be ascribed to the greater areal density of the adsorbed gemini IM-SAILs at the ALI and stronger hydrophobic interactions between LCs and gemini IM-SAIL molecules with longer chains. These results indicate that the interactions between the double alkyl chains of the gemini surfactants and LCs play a critical role in the orientational alignment of LCs. 3.1.2 Effect of gemini surfactants with different spacer length on the orientation

of 5CB. In order to investigate the relationship between gemini IM-SAILs with different spacer length and the anchoring of LCs at the ALI, we used [C12-s-C12im]Br2 (s=2, 4, 6, 10) aqueous solution to decorate the LC interface. Fig. 3 reveals that a bright-to-dark shift in the LCs optical response occurred with the increment of [C12-s-C12im]Br2 (s=2, 4, 6, 10) concentration. The Cthreshold of [C12-2-C12im]Br2, [C12-4-C12im]Br2, [C12-6-C12im]Br2 and [C12-10-C12im]Br2 is 0.5 µM, 0.8 µM, 1 µM and 5 µM, respectively. This observation implies that by comparison to gemini IM-SAILs with a longer spacer, those with a shorter spacer are more likely to form a stable monolayer at the ALI and trigger the planar-to-homeotropic orientational transition of LCs.

Fig. 3 POM images of 5CB at the ALI in the presence of [C12-s-C12im]Br2 (s=2, 4, 6, 10) aqueous solutions with various concentration.

As reported [34,35], when the number of carbon atoms in the spacer of gemini surfactants is in the range of 2-10, surface area per amphiphile at the air/water interface increases rapidly with the elongation of the spacer. At this point, the spacer in contact with the aqueous phase can stretch along the air/water interface (illustrated in Fig. 4a). However, once the number of carbon atoms in the spacer exceeds 10, the

headgroups become closer to each other and surface area per gemini amphiphile in fact decreases upon increasing the spacer length. The probable reason is that the longer spacer is too hydrophobic to remain at the air/aqueous interface and adopts a folded “wicket-like conformation” (demonstrated in Fig. 4b) [36,37]. For [C12-s-C12im]Br2 (s=2, 4, 6, 10), it is found that the gemini SAILs with shorter spacers are easier to adsorb onto the ALI and trigger the planar-to-homeotropic orientational transition of LCs. We speculate that surface area of each SAIL molecule increases together with an extension of spacer length and their spacer would stretch at the ALI. Here [C12-2-C12im]Br2 and [C12-10-C12im]Br2 are taken as examples. Schematic illustrations of LC orientation at the ALI in the presence of gemini IM-SAILs in aqueous solution are shown in Fig. 4c and 4d. A larger distance exists between the hydrophobic chains of each [C12-10-C12im]Br2 molecule and it reduces the surfactant tail-5CB interaction to a great enough degree to switch the anchoring of LCs from the planar to homeotropic state.

Fig. 4 (a, b) Two schematic representations of gemini surfactants at the air/aqueous interface [34]; (c, d) Illustrations of the LC alignment at the ALI upon addition of [C12-2-C12im]Br2 and [C12-10-C12im]Br2 aqueous solutions, respectively.

3.1.3 Effect of gemini surfactants with different symmetry on the orientation of

5CB. In this section, three gemini QASaa with the same headgroup, spacer length, and hydrophobic units yet with different symmetry (12-2-12, 14-2-10 and 16-2-8; Fig. 1c), were added to the ALI. It can be seen that the optical appearance of the LCs could undergo a bright-to-dark transformation (Fig. S2). Their Cthreshold are all about 2.5 µM, which suggests that they possess equivalent surfactant tail-5CB effect [8] to drive the LCs orientation at the ALI from the planar to homeotropic state. It follows that the symmetry of gemini surfactants has almost no influence on the alignment of LCs at the fluid interface. Conversely, previous reports [38] have shown that quaternary ammonium salt-type gemini surfactant molecules formed more closely packed arrays at the air/water interface with the increase of dissymmetry. Combining the above results, we can infer that the adsorption behavior of the gemini surfactants at the ALI is distinct from that at the air/water interface. 3.1.4 Effect of gemini surfactants with different headgroups on the orientation of 5CB. The Cthreshold values of [C12-2-C12im]Br2 (0.5 µM, Fig. 2) and 12-2-12 (2.5 µM, Fig. S2), we can explore the optical response of LCs at the ALI induced by gemini surfactants

with

different

headgroups.

Evidently,

compared

to

12-2-12,

[C12-2-C12im]Br2 is more inclined to form a surfactant monolayer at the ALI and trigger variation in the orientation of LCs. This may be linked to the weaker electrostatic repulsion between [C12-2-C12im]Br2 molecules due to the electron delocalization of imidazolium groups, Which encourages the surfactant molecules to pack more closely at the ALI. 3.1.5 Effect of the number of surfactant aliphatic chains on the orientation of 5CB. As a control experiment, the optical behavior of LCs at the ALI after incubation with DTAB (the monomer of 12-2-12) aqueous solution was investigated. It was found that the Cthreshold of DTAB, 0.02 mM (Fig. S3), is far higher than that of 12-2-12 (2.5 µM). This observation suggests that gemini surfactants are readily adsorbed onto

the ALI and form stable monolayers, in contrast with their monomer, which is in agreement with our earlier report [28]. The reason can be attributed to enhancement of the intramolecular hydrophobic interaction, resulting from the close connection between the two hydrophobic chains. 3.2 Imaging interactions of BSA with a series of gemini surfactants at the ALI. Herein, surfactant aqueous solutions were initially used to immerse 5CB to form stable monolayers at the ALI. Then the optical response of LCs was observed after equivoluminal protein aqueous solutions were added onto the LC interface. 3.2.1 Imaging Interactions of BSA and gemini IM-SAILs with different alkyl chains Here the concentration of gemini IM-SAILs was 0.01 mM, at which a stable monolayer could be formed at the ALI. Fig. 5a shows the evolution of the bright area coverage ratio (Br (%)) in LC images, when 0.2 mg/mL BSA was added onto ALIs pre-decorated with [C10-2-C10im]Br2, [C12-2-C12im]Br2 and [C16-2-C16im]Br2 aqueous solutions. It is noted that Br=100% of LC images is reached at 5 min and 30 min for [C10-2-C10im]Br2-BSA and [C12-2-C12im]Br2-BSA systems, respectively, while for [C16-2-C16im]Br2-BSA, the LC images only partly turned bright at 80 min. We speculate that BSA molecules adsorb onto the interfaces decorated with gemini IM-SAILs mainly through electrostatic interaction. This would imply that the desorption rates of the gemini IM-SAILs at the ALI would reduce with the increase of their tail length. Therefore, with an extension of the alkyl chain, the surfactant monolayers will be more stable and less prone to being disturbed by the electrostatic interaction between BSA and gemini IM-SAILs. Consequently, as was observed for the [C16-2-C16im]Br2-decorated aqueous/LC interface, BSA cannot induce the orientation of LC to completely transform from homeotropic to planar state even after 80 min. 3.2.2 Imaging interactions of BSA and gemini IM-SAILs with different spacer length Bright area to entire square area ratios Br(%) in the LC optical images as a

function of time after BSA was added to an ALI pre-decorated with [C12-s-C12im]Br2 (s=2, 4, 6, 10) aqueous solution, are demonstrated in Fig. 5b. The response times for the LC images to change entirely from dark to bright for [C12-10-C12im]Br2, [C12-6-C12im]Br2, [C12-2-C12im]Br2 and [C12-4-C12im]Br2 were 10 min, 15 min, 20 min and 30 min, respectively. To investigate the interaction mechanism, we calculated the electrostatic potential of the cations in the gemini IM-SAILs with Gaussian 09 [39]. Fig. 6 illustrates that the electropositivity of [C12-s-C12im]Br2 (s=2, 4, 6, 10) follows the order: [C12-2-C12im]2+ > [C12-4-C12im]2+ > [C12-6-C12im]2+ > [C12-10-C12im]2+. Previous reports have also shown that gemini surfactants with shorter spacers possess higher charge density [21,22]. Therefore, the sequence of electrostatic interaction between BSA and [C12-s-C12im]Br2 (s=2, 4, 6, 10) is in line with the order of electropositivity for the cations of surfactants. Furthermore, along with the values of s changing from 2 to 10 for [C12-s-C12im]Br2, the hydrophobicity of the spacer is enhanced, which suggests that the hydrophobic interaction between BSA and the spacers of gemini IM-SAILs increases in the following order: [C12-2-C12im]Br2 < [C12-4-C12im]Br2 < [C12-6-C12im]Br2 < [C12-10-C12im]Br2. Thus, we can infer that for [C12-s-C12im]Br2 (s=2, 4, 6, 10), hydrophobic interaction (between BSA and the spacers of the gemini IM-SAILs) and electrostatic interaction both play a major role in their interaction process. 100

100

80

80

[C10-2-C10im]Br2+ BSA

[C12-2-C12im]Br2+ BSA

[C12-2-C12im]Br2+ BSA

Br (%)

60

60

40

Br (%)

[C16-2-C16im]Br2+ BSA

a

20

[C12-4-C12im]Br2+ BSA [C12-6-C12im]Br2+ BSA

40

[C12-10-C12im]Br2+ BSA

20

0

b

0

0

10

20

30

40

50

Time (min)

60

70

80

0

10

20

30

40

Time (min)

50

60

Fig. 5 Bright area coverage ratio (Br (%)) with time-dependence in the POM images of LCs after addition of 0.2 mg/mL BSA onto the (a) 0.01 mM [Cn-2-Cn im]Br2 (n =10, 12, 16), (b) 0.01 mM

[C12-s-C12im]Br2 (s=2, 4, 6, 10)-decorated LC interfaces.

Fig. 6 Electrostatic potential, in Hartrees, calculated at the 0.001 electrons/bohr3 isodensity surfaces of (a) [C12-2-C12im]2+, (b) [C12-4-C12im]2+, (c) [C12-6-C12im]2+ and (d) [C12-10-C12im]2+ by Gaussian 09 at the level of B3LYP/6-31G.

3.2.3 Imaging interaction difference for gemini surfactants and their monomers with BSA Single-chained surfactants ([C12mim]Br and DTAB) and gemini surfactants ([C12-2-C12im]Br2 and 12-2-12) were chosen as study subjects, with concentration held constant at 0.05 mM, at which stable monolayers can be formed. Based on this, discrepancy in the interactions between them and BSA at the ALI was explored. Fig. 7 shows the time-dependent graphs of Br(%) in the LC optical appearances. It can be seen that the response rate of the LC images obtained from slopes of the lines was accelerated. The response time needed for the optical texture to turn from dark (Br=0%) to entirely bright (Br=100%) state was shortened with the increase of BSA concentration. In addition, the optical appearance of 5CB became completely bright, which indicates the monolayers were disturbed and the orientational transition of the LCs from homeotropic to planar state occurred, approximately corresponding to 0.01

mg/mL BSA for the single-chained surfactants (Fig. 7a and 7c) and 0.5 mg/mL for the gemini surfactants (Fig. 7b and 7d), respectively. We infer that that in contact with BSA, the monolayers formed by gemini surfactants at the ALI are more stable than those of the single-chained surfactants since the desorption rate constant of gemini surfactants is much lower than the single-chained surfactants at the ALI. Moreover, when the concentration of BSA is ≤0.05 mg/mL in the presence of gemini surfactants, or ≤0.005 mg/mL for the relevant monomers, the optical appearance of 5CB only partly changed to bright or even unchanged, indicative of a transition from homeotropic to tilted state or invariableness for the orientation of LCs. 100

Br (%)

0.001 mg/mL 0.005 mg/mL 0.01 mg/mL 0.02 mg/mL

a

80 60 40

80

Br (%)

100

20

0.01 mg/mL 0.05 mg/mL 0.5 mg/mL 1 mg/mL

b

60 40 20

0

0 0

10

20

30

40

Time (min)

50

60

0

10

20

30

40

Time (min)

50

60

100 100

0.001 mg/mL 0.005 mg/mL 0.01 mg/mL 0.02 mg/mL

60

c

40 20

80

Br (%)

Br (%)

80

0.01 mg/mL 0.05 mg/mL 0.5 mg/mL 1 mg/mL

60

d

40 20

0

0

0

10

20

30

40

Time (min)

50

60

0

10

20

30

40

Time (min)

50

60

Fig. 7 Time-dependent bright area coverage ratio of LC optical images after adding different concentrations of BSA onto ALI decorated with 0.05 mM [C12 mim]Br (a), [C12-2-C12im]Br2 (b), DTAB (c) and 12-2-12 (d).

3.3 Imaging interactions of gemini surfactants with trypsin or lysozyme As above, we monitored alignments of the LCs at the ALI decorated with

positively charged single-chained or gemini surfactants in the presence of negatively charged BSA. In accordance with previous reports[5], we suppose that the electrostatic attractive interaction between these surfactants and BSA plays a critical role in triggering the reorientation of 5CB molecules at the fluid interface. Therefore, we expected to find that electrostatic repulsion between surfactants and protein may not change the alignment of the LCs. To test our hypothesis, interactions between another two proteins with positive charge in neutral pH water, namely trypsin and lysozyme (with pIs of 10.5 [40] and 11.4 [23], respectively) and single-chained or gemini surfactants at the ALI were explored. Here the concentration of surfactants was still fixed at 0.05 mM. 3.3.1 Imaging interactions of gemini surfactants with trypsin For

comparison,

the

interactions

of

0.05

mM

[C12mim]Br,

DTAB,

[C12-2-C12im]Br2 and 12-2-12 aqueous solution with trypsin at the ALI were investigated. Fig. 8 shows the time-dependent bright area coverage ratio of LC images after addition of different concentrations of trypsin onto the LC interfaces pre-incubated with various surfactant aqueous solutions. Unexpectedly, the optical appearance of 5CB varied from dark to bright when the single-chained surfactants came into contact with relatively high concentrations of trypsin (Fig. 8a and 8c). This observation demonstrates that transformation of the LCs alignment from the homeotropic to planar state is due to the disturbance of the surfactant monolayer at the ALI. As reported, interactions of proteins with surfactants mainly proceed through electrostatic interactions and hydrophobic interactions [19-24]. Therefore, we assume that the disturbance of the single-chained surfactant monolayer is on account of the hydrophobic interactions between surfactant and trypsin. However, unlike the optical appearances of single-chained surfactants at the aqueous solution/LC interface, the optical images of 5CB in all cases remained dark for the [C12-2-C12im]Br2-decorated LC interface in the presence of trypsin (0.01-1 mg/mL) (Fig. 8b), demonstrating that addition of trypsin cannot change the alignment of LCs at the interface. Similar phenomena were observed with 12-2-12 in contact with the trypsin aqueous solutions (0.01-0.5 mg/mL) (Fig. 8d). Evidently, for the

gemini surfactant-trypsin systems, the experimental results are consistent with our initial speculations, while for the single-chained surfactants the case is to the contrary. A possible reason is that the hydrophobic interaction between gemini surfactant tails and 5CB is stronger than that of gemini surfactants with trypsin, and thus it is difficult to destroy the surfactant monolayers. Particularly interesting is that for some systems, the bright area coverage ratio of LCs primarily increased and then decreased over time, e.g. 0.1 mg/mL trypsin/0.05 mM DTAB (Fig. 8a), 0.1mg/mL (0.5 mg/mL) trypsin/0.05 mM [C12mim]Br (Fig. 8c) and 1 mg/mL trypsin/0.05 mM 12-2-12 systems (Fig. 8d). It can be attributed to that the electrostatic repulsions between these surfactants and trypsin may lead to the reorientation of 5CB molecules over time at the ALIs, which makes the optical appearance of LCs less stable.

80

80

60

60

40

0.01 mg/mL 0.1 mg/mL 0.5 mg/mL 1 mg/mL

a

20

Br (%)

100

Br (%)

100

b

40 20

0

0

0

10

20

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Fig. 8 Time-dependent bright area coverage ratio of LC optical images after adding different concentrations of trypsin onto ALI decorated with 0.05 mM [C12mim]Br (a), [C12-2-C12im]Br2 (b), DTAB (c), 12-2-12 (d).

3.3.2 Imaging interactions of gemini surfactants with lysozyme

The interaction of Lysozyme, with positive charge in neutral water, with 0.05 mM [C12mim]Br and [C12-2-C12im]Br2 at the ALI was also chosen for study. After adding different concentrations of lysozyme onto the LC interfaces pre-incubated with [C12mim]Br and [C12-2-C12im]Br2 aqueous solution, we could obtain the plots of bright area coverage ratios in the LC optical images over time (Fig. 9). Similarly, as for the [C12mim]Br-decorated aqueous/LC interface, it is observed that the optical appearance of 5CB underwent a dark-to-bright complete transformation after in contact with lysozyme in the concentration range of 0.001~0.05 mg/mL (Fig. 9a), suggesting the homeotropic-to-planar reorientation occurred at the ALI. Yet, for gemini surfactant [C12-2-C12im]Br2 (Fig. 9b), the optical appearance of LCs was unchanged, which can be ascribed to the stronger interaction of [C12-2-C12im]Br2 tails with 5CB than that with lysozyme. 100

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Fig. 9 Time-dependent bright area coverage ratio of the POM images of LC at the ALI decorated with 0.05 mM [C12mim]Br (a), [C12-2-C12im]Br2 (b) in the presence of different concentrations of lysozyme.

3.4 Mechanisms of interactions between gemini surfactants and proteins at the ALI To sum up the

results of Figs. 7-9, we

can conclude that the

[C12-2-C12im]Br2-decorated LC platform can be used to distinguish the proteins with different surface electrical properties, such as the proteins with negatively (BSA) and positively charge (lysozyme or trypsin) in neutral water medium. The mechanisms of interaction between gemini surfactants and these three proteins are speculated upon

and illustrated in Fig. 10. We suggest that the orientation of LCs depends on the competition of the interaction effect of [C12-2-C12im]Br2 tails with 5CB and [C12-2-C12im]Br2 with proteins at the ALI. When BSA with negative charge is added to the interface pre-incubated by [C12-2-C12im]Br2, the interactions between BSA and gemini surfactants (combination of electrostatic and hydrophobic interactions) are far stronger than those of [C12-2-C12im]Br2 tails with 5CB, which then triggers the homeotropic-to-planar reorientation of the LCs at the fluid interface. Yet, for lysozyme or trypsin with positive charge, the electrostatic repulsion between proteins and gemini surfactant inhibits the adsorption of protein at the ALI. Herein, the stronger hydrophobic effect of [C12-2-C12im]Br2 tails with 5CB plays a decisive role, resulting in no change of the LCs orientation.

Fig. 10 Illustration of the LC alignment at the ALI pre-incubated by [C12-2-C12im]Br2 aqueous solution in the presence of proteins.

Conclusions In earlier studies, surfactant-laden LC-based sensing platforms were widely constructed [6-16], however, they were mostly limited to single-chained surfactants, with gemini surfactants seldom involved. In this work, cationic gemini surfactants (viz. gemini IM-SAILs and gemini QASaa) with diverse chemical structures were

synthesized and applied to decorate the ALI. Compared to the self-assembled monolayers constructed by their monomers at fluid interface, the gemini surfactant monolayers were more stable due to the stronger surfactant tail-5CB effect. At present although there are some reports on the interactions of surfactants with proteins in the bulk phase and at the air/water interface [19-24], the corresponding investigations at the ALI have not been mentioned. In this work, electrostatic attractive interaction between gemini IM-SAILs and BSA can destroy the monolayers formed by the former at the ALI and further evoke the reorganization of LCs. However, electrostatic repulsion between the cationic gemini IM-SAILs and lysozyme or trypsin cannot change the anchoring of LC at the fluid interface. Based on the above results, LC-based sensing platforms decorated with cationic gemini surfactants could be established to identify proteins with different charges, e.g. negative-charged BSA and positive-charged lysozyme or trypsinin in the neutral aqueous solution. This work presents the feasibility of building a simple, convenient and inexpensive LC-based sensor to distinguish proteins.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21373128) and Scientific and Technological Projects of Shandong Province of China (No. 2014GSF117001).

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Supporting Information More details about Synthesis of gemini IM-SAILs, gemini QASaa and [C12mim]Br.

Graphical Abstract