lcrprt

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Detection of Biomolecules using Surface-

driven Ordering Transitions in Liquid

Crystal

By

Nishtha Agarwal

Summer Project under the guidance of

Dr. Santanu Kumar Pal

INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH MOHALI,

SECTOR- 81, S. A. S NAGAR, MANAULI PO, PUNJAB- 140306

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CERTIFICATE

This is to certify that Nishtha Agarwal of IISER Mohali persuading Integrated

MS in 5th Semester has successfully completed her summer internship under

the guidance of Dr. Santanu Kumar Pal on “Detection of Biomolecules using

Surface-driven Ordering Transitions in Liquid Crystal”.

Dr S.K. Pal

Assistant Professor, Chemistry

IISER Mohali

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DECLARATION

I hereby declare that the entire work embodied in this report is the result of the

experimental investigations carried out by me at IISER Mohali, under the

guidance and supervision of Dr. Santanu Kumar Pal.

Dr S.K. Pal Nishtha Agarwal

Assistant Professor, Chemistry MS09091

IISER Mohali IISER Mohali

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Detection of Biomolecules using Surface-driven

Ordering Transitions using Liquid Crystals

Nishtha Agarwal

Indian Institute of Science Education and Research Mohali,

Mohali, India

E-mail: [email protected]

Abstract

Liquid Crystals materials are a phase of matter with orientational order and some or no

positional order. They are between the crystalline solids and isotropic liquids. They are found

to be anisotropic in nature and hence show optical properties like birefringence etc. By effect

of external stimuli such as surface preparations, liquid-crystals can be aligned in a

homeotropic orientation. Bio-molecules can cause a change in this orientation by interacting

with the liquid crystals and this change can be observed in polarizing microscope. The

change can help us to identify the bio-molecule. The project was to use these surface driven

transitions to identify Lipopolysaccharide, an endotoxin found in bacterias.

Key-words used: Liquid Crystal, biosensors, bio-medical, Lipopolysaccharide, birefringence,

SDS, nematic phase, smectic phase, 5CB, E7, LC physical gels

Introduction

Liquid Crystal materials consist of anisotropic molecules that self-assemble into phases that

maintain some degree of orientational order and sometimes some positional order also. These

phases exist between the conventional crystalline phase and the isotropic liquid phase.

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Another name for them is Mesomorphic Phases (mesomorphic: of intermediate form). Most

liquid crystal compounds exhibit polymorphism or a condition where more than one phase is

observed in the liquid crystalline state. The term mesophase is used to describe the "sub-

phases" of liquid crystal materials. Mesophases are formed by changing the amount of order

in the sample, either by imposing order in only one or two dimensions, or by allowing the

molecules to have a degree of translational motion.

Characteristic properties of Liquid Crystals

Liquid crystal materials generally have several common characteristics. Among these are rod-

like molecular structures, rigidness of the long axis, and strong dipoles and/or easily

polarizable substituents.

The three main paramteres to describe liquid crystalline structure are:

Orientational Order: The distinguishing characteristic of the liquid crystalline state is the

tendency of the molecules (mesogens) to point along a common axis, called the director on

the long range basis. To quantify just how much order is present in a material, an order

parameter (S) is defined. It is generally defined based on the average of the second Legendre

polynomial, though higher order averages can yield additional information about molecular

ordering. Typical values for the order parameter of a liquid crystal range between 0.3 and 0.9,

with the exact value a function of temperature, as a result of kinetic molecular motion.

Positional Order: It refers to the extent to which an average molecule or group of molecules

shows translational symmetry (as crystalline material shows).

Bond Orientational Order: It describes a line joining the centers of nearest-neighbour

molecules without requiring a regular spacing along that line. Thus, a relatively long-range

order with respect to the line of centers but only short range positional order along that line

From an application point of view, the most useful properties of the liquid crystalline phase

are: anisotropy, birefringence, iridescence, selective absorption of the plane polarized light

and strong influence in magnetic and electric fields.

Optical properties and characterization

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Birefringence is an optical property used to further characterize LCs. This is present due to

their anisotropic nature. That is, they demonstrate double refraction (having two indices of

refraction). Thus, presence of LCs can be easily judged using two cross polarizers.

And by effect of external stimuli such as surface preparations, the molecules align themselves

in a specific direction. The resulting distortion can then be addressed by optical methods and

any changes in the original alignment are correlated with the identity of the contaminant.

Classification of Liquid Crystals

There are two generic classes of liquid crystals: those whose transitions are driven by thermal

processes, known as thermotropics, and those strongly influenced by solvents, known as

lyotropics. There is also a class called Metallotropic. Metallotropic LCs are composed of

both organic and inorganic molecules; their LC transition depends not only on temperature

and concentration, but also on the inorganic-organic composition ratio.

Thermotropic Liquid Crystals

Thermotropic phases are those that occur in a certain temperature range. If the temperature

rise is too high, thermal motion will destroy the delicate cooperative ordering of the LC

phase, pushing the material into a conventional isotropic liquid phase. At too low

temperature, most LC materials will form a conventional crystal

Lyotropic liquid crystal

A lyotropic liquid crystal consists of two or more components that exhibit liquid-crystalline

properties in certain concentration ranges. Lyotropic liquid crystal phases are formed by

amphiphilic molecules. When these are dissolved in an appropriate solvent they self-assemble

to form micelles like structures.The content of water or other solvent molecules changes the

self-assembled structures. At higher concentration, the assemblies will become ordered.

In either case, the interactions between the anisotropic molecules promote orientational and

sometimes positional order in an otherwise fluid phase.

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Fig1 Some substances exists in intermediate phases, called “Mesophases”. Tm and Tc are the

melting and clearing temperature, respectively.

There are many types of liquid crystal states, depending upon the amount of order in the

material. Broadly they are classified in Nematic phase which has only orientational order and

smectic phase which has certain positional order also.

Mesophases of the liquid crystalline structure

Nematic phase: In a nematic phase, the molecules have no positional order, but they self-

align to have long-range directional order with their long axes roughly parallel. Thus, the

molecules are free to flow and their center of mass positions are randomly distributed as in a

liquid, but still maintain their long-range directional order. Most nematics are uniaxial: they

have one axis that is longer and preferred, with the other two being equivalent (can be

approximated as cylinders or rods). However, some liquid crystals are biaxial nematics,

meaning that in addition to orienting their long axis, they also orient along a secondary axis.

Smectic phase: Smectic phases have further degrees of order compared to the nematic phase.

In the simple smectic phase, the molecules order into layers, thus positionally ordered along

one direction, with the layer parallel or tilted to the director. There are many other smectic

phases which have long range order within the layers. Smectic phases can also be formed by

chiral molecules, leading to chiral smectic phases.

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Cholesteric phase: cholesteric phase, also called the chiral liquid crystals, are formed by

doping a chiral agent in lc. Hence, the director is no longer fixed in one direction, but

possesses a periodically helical rotation along an axis.

Based on the geometrically structure of LC molecules, LCs can be further divided into three

classes, including the (a) calimitic, (b) discotic, (c) lath-like molecules.

Applications of the Liquid Crystals

With the maturation of the information display field, liquid-crystal materials research is

undergoing a modern-day renaissance. Apart from the extensive use of LC in the electronics

industry as display devices, they have also found recent biomedical applications. Three

primary areas of application are presented: Devices and configurations based on liquid-

crystal materials are being developed for spectroscopy, imaging and microscopy, leading to

new techniques for optically probing biological systems. Liquid-crystal polymers are starting

to be used in biomimicking colour-producing structures, lenses and muscle-like actuators.

Biosensors fabricated with liquid-crystal materials can allow label-free observations of

biological phenomena, the third one being the main aim of this project.

Liquid crystal tunable filters are used as electro optical devices, e.g. in hyper spectral imaging

Thermotropic chiral LCs whose pitch varies strongly with temperature can be used as crude

liquid crystal thermometers, since the color of the material will change as the pitch is

changed.

Spectroscopy and Imaging

The switchable electro-optical properties of liquid crystal materials for optical components

make them ideal for the development of biomedical devices. Liquid-crystal tunable filters and

spatial light modulators build on various material configurations and are opening new

pathways for efficient, low-cost optical components and integrated systems. They allow rapid

optical screening of cells, tissues etc.

Materials for Bio-mimicry

Liquid-crystal materials can mimic structural color and tunable adaptive optics of natural

systems, and can be used for innovative actuators, micro-electromechanical (MEM) and

biological micro-electromechanical (bio-MEM) devices, such as artificial muscles. In the

simplest forms, actuator devices can be used as sensors or to induce fluid flow; on more

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complex scales, liquid-crystal material actuators are envisaged as artificial muscles capable of

carrying out complex tasks.

Bio-Sensors- Rapid diagnostics

LC materials, with their birefringent properties and extreme sensitivity to surface

interactions, are poised to play an important role in these devices. Optical sensors using

liquid-crystal materials could eliminate the need for markers or tags, as the liquid-crystal

molecules act to enhance the optical appearance of signals of a biological process or

structure. In observing such biosensors, label-free observations of enzymatic action and

molecular assemblies are made by detecting an optical change in the transmittance through a

liquid-crystal material.

The self assembly of surfactant and phospholipids at the liquid-crystal interface largely

depends on the surfactant tail length and molecular branching. The presence of biological

processed can also be investigated within bulk liquid-crystal films. Here, director distortions

vary greatly in the presence of a binding event. Optical detection of bacteria and viruses with

liquid-crystal materials has great potential for diagnostics.

New areas of application in the realms of biology and medicine are stimulating innovation in

basic and applied research into these materials.

Liquid crystal physical gels

Gelation of organic fluids and water has attracted a great deal of attention because it yields

elastic soft materials, which can be used as functional materials in a variety of fields. Liquid-

crystalline (LC) physical gels are a new class of dynamically functional materials comprising

3D fibrous aggregates of low-molecularweight compounds. The molecules that induce

gelation are called ‘‘gelators. Liquid-crystalline physical gels are obtained by the self-

assembly of fibrous solid networks of gelators in liquid crystals. Liquid crystals become soft

solids by gelation, keeping their stimuli-responsive properties. The formation of anisotropic

phase-separated structures leads to the induction of new functions and the enhancement of the

properties. The LC physical gels are normally formed by thermal processes, in which two

independent transitions, the sol–gel transition of a gelator and the isotropic–anisotropic

transition of a liquid crystal, are observed.

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Fig 2 Schematic representation of (a) physical gel and (b) liquid-crystal physical gel

EXPERIMENT

The main aim of the project is to study the application of liquid crystals as biosensors to

detect biomolecules namely, Sodium Dodecyl Sulphate (SDS) and Lipopolysaccharide (LPS)

Lipopolysaccharides (LPS), also known as lipoglycans, are large molecules consisting of

a lipid and a polysaccharide joined by a covalent bond; they are found in the outer

membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune

responses in animals. It acts as a pyrogen and a dose of as low as 1µg/kg induces shock in

human. Higher quantities can even lead to death and thus its detection in an easy, convenient

manner is very crucial.

LCs biosensors provide an easy, label free method to detect very low concentrations of

contaminants.

Principle involved

At the pure liquid-crystal/ aqueous interface, the liquid-crystal molecules tend to align planar

to the surface. The introduction of contaminants (molecules for detection) induces an

orientational change of the liquid-crystal material to a homeotropic alignment. During this

transition, the optical appearance of the biosensor will change when viewed between crossed

polarizers and this is correlated with either the identity of the contaminant and/or its

concentration.

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Thermotropic calamitic liquid crystal materials were used throughout the experimental work.

Mainly 5CB and E7 were used which are described below.

5CB - 4-Cyano-4'-pentylbiphenyl

Fig3 Structure of 5CB

5CB  is a nematic liquid crystal with the chemical formula C18H19N. It frequently goes by the

common name 5CB. 5CB was first synthesized by George William Gray, Ken Harrison, and

J.A. Nash at the University of Hull in 1972 and at the time it was the first member of the

cyanobiphenyls. The liquid crystal was discovered with the specific intention of using them

in liquid crystal displays but now it has many more applications because it exhibits liquid

crystallinity in the nematic phase between 24ºC and 35ºC and is often the reagent choice in

simple optical studies . The molecule is about 20 Å long. The liquid crystal 5CB undergoes a

phase transition from a crystalline state to a nematic state at 18 °C and it goes from a nematic

to an isotropic state at 35 °C.

E7

The nematic liquid crystal, E7 is a compound of four species of pure liquid crystals: K15(4-

pentyl-4 cyanobiphenyl 1), K21( 4-heptyl-4 -cyanobiphenyl), M24 (4-hexyloxy-4

cyanobiphenyl), and T15(4-pentyl-4 -terphenyl).  The mixing ratios of these pure liquid

crystals are, respectively, 47, 25, 18, 10 wt%. E7 is a nematic liquid crystal(NLC) at room

temperature (~20ºC). The transition temperature from the nematic to the isotropic state is

about 60ºC. The range of temperatures at which E7 is NLC is from -10 ºC to 60.5 ºC.

Fig 4 Structures of the components of E7

Experimental Work

Detection of SDS

K15K21M24T15

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Surface Preparations: Slides were first cleaned using acidic piranha solution and

subsequently washed with ethanol.

After drying them overnight some of them were covered with OTS

(Octadecyltrichlorosilane), they were washed with chloroform and kept for

drying.

SDS detection: Gold grids (20µm) were put carefully on OTS covered slides and

about 2- 3 µl of E7 (LC) was put using micro-syringe and rest was removed to form

uniform layer of liquid crystals.

After drying, it was observed under polarizing microscope (crossed polarizers)

Firstly, water was added and then SDS was added to the solution.

Changes in the orientation were observed and noted.

Observations : The slides were black due to homeotropic alignment of the liquid crystals. On

addition of water, the alignment was disturbed due to hydrophobic-hydrophilic interactions

and thus it became bright.

On addition of SDS, it again started to become black and finally after addition of about 800µl

SDS(.2mM) it became black.

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Fig5 (a) The figure shows the black region which is due to homeotropic alignment of the

liquid crystal put on gold grid (20μm). Fig5 (b) The figure shows the section of the grid in

the same position. Fig5 (c) The figure shows change in alignment of the liquid crystals on

addition of water. Fig5 (d) Liquid Crystals again became homeotropic on addition of SDS

solution (0.2 mM), and thus the slide again started becoming black.

Conclusion: This proves the detection of SDS as the orientation of liquid crystals changes as

observed under polarized microscope.

LC GELS preparation

Gel Preparation: 15% w/v (15 µg polystyrene particles and 100 µl E7) were taken in

an Eppendorf tube and were sonicated at a temperature of about 80 °C for about 5 hrs

with continuous shaking in vortex every 5 minutes. It was kept for slow and uniform

cooling.

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Fabrication of LC gel device: Spacers were placed on all the four sides of a clean

glass slide. LC gel was placed in the center and it was kept in an over preset to the

temperature of about 100ºC.

Then after 8-10 mins, each slide was covered it another glass slide and sealed

using paper binders and kept in over for another 5 mins. Then it was switched off

and slide was kept for slow and uniform cooling.

Observations: Initially the gels were only observed under low magnification crossed

polarizers. LC domains were observed and black region of polystyrene particles was seen

under the microscope.

Fig 6 (a) LC physical gels prepared on glass slides. (b) LC gels observed under crossed

polarizers in the microscope. Polystyrene particles are expelled out of the nematic phase

forming LC domains.

Extraction of Lipopolysaccharide:

Preparation of e-coli culture

A batch of 200 ml LB media was inoculated by 2% of e-coli culture (top-ten

Strain) which was incubated overnight. This batch was then again incubated at

170 rpm at 37 °C for 18 hours.

Then cultured e-coli was centrifuged at 12000 rpm at 4 °C for 25 minutes. The

supernatant was discarded and the debris part was then washed with PBS and

stored at -80°C.

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Extraction of lipopolysaccharide

The stored e-coli batch was centrifuged and washed with saline and thrice with

acetone (300 ml of acetone for 10 gm of bacteria). Autome dried powder(acetone

free) was created by drying the liquid paste formed by centrifugation along the

walls of a beaker at room temp.

5 gm of acetone dried bacterial + 90 ml of distilled H2O (pretreated to 70 °C) and

100 ml of 90% phenol at 65°C - 80°C was added to it and vigorously shaked and

the mixture was kept at 65°C in water bath.

Then this was cooled to 10°C in an ice bath and the resulting emulsion was

centrifuged at 3000 rpm for 45 minutes at 4°C which resulted in formation of 3

layers.

Top layer – aquous layer containing lps

Bottom layer - phenol

Insoluble cell mass at the bottom and white debris in the separation of the liquid

layers

The top most water layer was carefully withdrawn while the phenol and insoluble

residue further extracted with another 90 ml of hot water as done before. The

combined water extract was dialyzed for 3 to 4 days against water to remove

phenol and small amounts of low molecular weight substances. The slightly

opalescent solution containing mainly LPS and RNA was concentrated to a

volume of about 50 ml and centrifuged at 3000 rpm for 10 minutes to remove

traces of insoluble material. The extract was then dissolved in 75 ml of water and

7.5 ml of 2% cetyl trimethyl ammonium bromide(c-TAB). The mixture was

stirred for 15 minutes at room temperature and then centrifuged for 30 minutes at

5000 rpm to remove precipitate RNA material slightly opalescent was stored at

4ºC.

NaCl was added to the solution to make the final concentration 0.19 M and 10

folds (800ml) of absolute ethanol was added and the mixture was allowed to stand

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for 2 hrs at 4°C to precipitate LPS. The precipitate was centrifuged and dissolved

in water. This was then freeze dried to give fluffy white powder of LPS.

It still contains some amount of protein which might hinder in the detection of lps, thus it will

be made free from protein using pronase treatment.

Conclusions:

Liquid Crystals provide label-free instruments for qualitative and quantitative detection of

bio-molecules by surface driven tranisitions in the orientation. This provides an easy to use,

and efficient biosensors which can detect contaminants in quantities as low as few ppm.

References :

[1] Colling, Peter J. and Hird Micheal. Introduction to Liquid Crystals, 1997, Taylor &

Francis.

[2] Woltman et al. Liquid-crystal materials find a new order in biomedical applications, 2007,

Nature Materials, 6, 929 – 938.

[3] Kato, Takashi et al. Liquid-crystalline physical gels, 2007, Chemical Society Reviews,

Volume 36, Number 12, 1845–2128.

[4] Jeffrey M. Brake, et al. Biomolecular Interactions at Phospholipid-Decorated Surfaces of

Liquid Crystals, 2003, Science 302, 2094.

[5] Rahul R. Shah et al. Principles for Measurement of Chemical Exposure Based on

Recognition-Driven Anchoring Transitions in Liquid Crystals, 2001, Science 293, 1296

[6] Takashi Kato, Norihiro Mizoshita, and Kenji Kishimoto . Functional Liquid-Crystalline Assemblies: Self-Organized Soft Materials (Review), 2006, Angew. Chem. Int. Ed., 45, 38.

[7] Roque et al. Bio-recognition and detection using Liquid crystals (Review), 2009, Elsevier, 25, 1 – 8.

[8] S- T. Wu and C- S. Wu. 1990, Physics Review A 42 , 2219 – 2227.

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Acknowledgement

Through this acknowledgment, I express our sincere gratitude to all those people who have

been associated with this assignment and have helped me with it and made it a worthwhile

experience. Firstly, I express my thanks to Dr. Santanu Kumar Pal who gave me this

opportunity to learn the subject in a practical approach and guided me and gave me his

valuable suggestions regarding the project report. Also, I extend my thanks to Dr Kaushik

and his team who have shared their opinions and experiences through which I received the

required information crucial for our report. Finally, I thank IISER Mohali for providing the

necessary infrastructure and help regarding my project.

Nishtha Agarwal

MS09091

IISER Mohali