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Summer Research Fellowship Programme of India's Science Academies

Investigation of spectral responses of FF dipeptide microrings

Dhanya Y Bharadwaj

Christ University, Hosur Main Road, Bengaluru 560029

Dr Ayan Banerjee

Indian Institute of Science Education and Research Kolkata, Campus Road, Mohanpur, West Bengal 741246

Abstract

'Self-assembly' of peptide molecules possesses widespread applications due to their photo-luminescent, conductive, ferro-electric, sensing, energy storing and wave guiding properties. One of the simplest dipeptides exhibiting these properties is diphenylalanine (FF) which is a core recognition motif of Alzheimer’s β-amyloid poly-peptide. In our experiment we employ a novel technique for the formation of FF microrings using water vapor bubbles formed due to the creation of hot spot in ‘thermo-optical tweezers system’. When the dispersion of Soft oxometalates (SOMs) which has high absorption at 1064nm wavelength is taken on a glass cover-slip and laser of wavelength 1064nm is introduced, a hot spot is created leading to formation of water bubble around it. Presence of temperature gradient on the surface of the bubble leads to gradient in surface tension which causes ‘Gibbs-Marangoni’ flow that result in influx of particles at the base of the bubble. Then the trapped bubble is translated using the microscope sample holder stage of the apparatus so that the nucleation site of the material is simultaneously translated generating continuous patterns. The process is continued to obtain desired pattern of trails which can be used for formation of micro-rings. When FF solution is dropcasted on the cover slip, the laser of wavelength 1064nm is again focused on the obtained trail which leads to formation of FF microring structures. Microrings thus obtained are of special interest as they are due to directed self assembly wherein it allows the accurate control of morphology, size and spatial location using laser induced micro bubble technique. When these microrings were excited at one end, circulation of light along the ring was observed which is clearly emphasizing on the wave guiding property of FF dipeptides. These microrings may be employed in the detection of Alzheimer’s disease by analyzing its spectral responses.

Keywords: self assembly, diphenylalanine, wave guide, optical tweezers

Abbreviations

Abbreviations
 CR Congo Red
 FF Diphenylalanine
 GIF Graded index fiber
NA  Nummerical aperture
 LIMBTLaser Induced Microbubble Technique 
 SHGSecond Harmonic Generation 
SOM Soft Oxo Metalates 
TE Transverse Electric 
 TEMTransverse electromagnetic 
TIR Total Internal Reflection 
TM Transvers magnetic 

INTRODUCTION

Background

Self-assembly is a process where a cluttered arrangement of previous components shapes a composed structure due to local interactions without an aid of external direction. 1 It is common and widely seen everywhere in nature and technology. Self assembly being an essential part of nanotechnology, self assembled nanomaterials have been predicted to show wide range applications in optic, electronic ,magnetic and other functional devices due to their photo-luminescent, conductive, ferro-electric, sensing, energy storing and wave guiding properties.  2  

Picture1.png
    Depiction of molecular arrangement of nanoparticles before and after self assembly process. It is very clear from the picture that the molecular arrangement attained organized structure from disorderdness due to some specific interactions.

    In the past decades, great efforts have been focused on self-assembly of peptide molecules owing to their structural simplicity, bio-compatibility, biodegradability, functional flexibility, chemical versatility, facile synthesis and widespread applications. 3  Assembly could be effectively achieved by hydrogen bonding, electrostatic, hydrophobic, and π–π stacking interactions. Such simplest peptide building block is diphenlyalalnine(FF), the core recognition motif of Alzheimer’s β-amyloid polypeptide. Over past few years extensive studies have been made on FF dipeptides due to their exceptional physical and chemical properties.

    Picture2.png
      Molecular structure of Diphenylalanine (FF) dipeptide.

      Due to their wide range applications, various methods have been administered for synthesis of FF dipeptides. Researchers have shown that the polymorphism of FF-based assembly can be influenced and easily controlled by the experimental conditions such as solvents, peptide concentrations, pH and temperature. However, because of the strong self-organizing tendency of the deposited materials, the production of a highly controllable arrangement of FF single crystals still faces many challenges, which surely limit their applications. Thus controlled synthesis of dipeptide synthesis can increase their applications in the field of sensing, optical switching and other biomedical fields.

      Among various morphology of FF such as microrods, microtubes, microrings, microwires, nanotubes, nanofibres, nanowires, nanoplates and nanovescile; FF microrings are of our interest due to their control in spatial location and size. In our project, we employ a novel technique for the formation of FF microrings using water vapor bubbles formed due to the creation of hot spot in ‘thermo-optical tweezers system. 4 In this method laser induced microbubbles leads to Gibbs marangoni convection which causes assembly of particles at the base of the bubble due to surface tension gradient in the bubble. The bubble thus trapped is translated using microscope sample holder stage to obtain desired pattern of trails. FF solution is then dropcasted on the trails to obtain FF microrings. This method is fast when compared to other methods used to obtain different morphology of FF described in literature section.

      These microrings exhibit wave-guiding properties both in cases of broadband white light and laser light. In case of broadband white light, only certain wavelength are guided resulting in Fano resonances characterized by asymmetric profile which has wide range application in optical switching, sensing etc.

      Presently, CR dye is widely used for detection of Alzheimer’s disease. In our case, the CR doped FF microrings show changes in both waveguiding characteristics and Fano asymmetric parameters. This might help in early detection of Alzheimer’s as well as being an affordable tool in the same.

      Objectives of the Research

      The main objective of this project is to investigate the spectral responses of FF dipeptides and their comparison with spectral response of Congo red doped FF dipeptides.

      Scope

      The directed self assembly of FF micro rings exhibit waveguiding poperties. When FF microrings are doped with dye called congo red which is currently used for detection of Alzheimers’s disease changes in their waveguiding characteristics and asymmetrical profile can be observed. In future this might have significant application in early detection of Alzheimers’s disease.

      LITERATURE REVIEW

      Self Assembly of FF dipeptides

      Study on obtaining different morphology of FF dipeptides:

      In different solvent and solute concentrations and other controllable factors can determine different morphological structures like microrods, microtubes, microrings, microwires, nanotubes, nanofibres, nanowires, nanolates and nanovesciles.

      Synthesis of inorganic nano materials:

      Inorganic nanostructured materials synthesized by using either of these complementary strategies: the “top-down” strategy or the “bottom-up”strategy. The first approach is essentially a “whittling” method, whereby a bulk material is reduced down to nanoscale objects. This approach offers precise control over the size and shape; however, the point-by-point or layer-by-layer processing makes this approach time-consuming. In contrast, the “bottom-up” approach involves constructing nanostructures one atom or molecular unit at a time by chemical synthesis and one unit at a time through self-assembly. This approach is simple and flexible, and the building blocks can be designed precisely to facilitate the assembly of nanostructures with obtainable features.

      Self assembly in inorganic nanomaterials:

      Self-assembly processes are critical for the “bottom-up” construction of nanostructures. Self-assembly enables the fabrication of three dimensional structures on the nano and micrometer scale and are commonplace in biology.

      Self assembly in dipeptides:

      Depending on the conformation and stereo-chemical configuration of their constituent amino acids, peptides exhibit different secondary structures, such as α helices and β sheets. The β-sheet structure has been studied intensively not only because of its important role in diseases related to peptide fibrillization, but also because of its application in the design and assembly of well-defined functional materials.

      Various molecules can be attached to peptides to affect their self-assembly properties and direct their assembly into particular desired structures. Such modified peptides are often termed “peptide conjugates”. Peptide conjugates can self-assemble into various well defined nanostructures.​​

      Methods for synthesis of dipeptides through self assembly:

      Vapour deposition method:

      Bingbing Sun and co workers have described procedures for self assembly of ultra long aligned peptide molecules and controlled rod nanostructured assembly of diphenylalanine in a capillary. ​​5​​

      Picture3.png
        Growth of an individual FF fiber inside a capillary starts within a few minutes after the beginning of the evaporation at room temperature. (a) Bright field images of the FF fiber. (b) Schematic diagram of the growth of FF fiber.

         

        With the evaporation of solvent, nucleation of the crystal occurred in the confined region, and the crystal grew continuously with a supply of molecules from the concentration gradient system inside the capillary. Based on the “Knudsen regime”, an ultralong aligned individual FF single crystal possessing an active optical waveguide property at macroscopic length scale could be obtained.

        Picture4.png
          Optical waveguiding of ultra long FF fiber. (a) Schematic of optical waveguidig in the fiber. (b) Optical images showing waveguiding property of FF fiber in the presence of white light and in the absence of it.

          Moreover, capillary is also an effective micro-device to investigate the disassembly process of the FF single crystals. This strategy has potentials to broaden the range of applications of aligned organic nanomaterials. In this method the FF molecules in HFP (Hexafluropropanal)/water solution doped with Rhodamine dye gives Rhodamine doped FF fibers which exhibit waveguiding property. This method also gives control on the number of fibers that can be obtained.

          Nanotubes:

          The self-assembly of cationic dipeptides is driven by hydrogen bonding and by π–π stacking interactions, as described by Reches and Gazit for the self-assembly of aromatic dipeptides into nanotubes.​3

          Amir Handelman and co workers 6 have focused on new specific functionality of ultrashort peptide nanotubes to guide light at fundamental and second-harmonic generation (SHG) frequency in horizontal and vertical peptide nanotube configurations. Conducted simulations and experimental data showed that these self assembled linear and nonlinear optical waveguides provide strong optical power confinement factor, demonstrate pronounced directionality of SHG and high conversion efficiency of SHG approximately equal to 10510^{-5}​​

          Nanovesicles:

          Qi Li and co- workers experimentally verifies that the nanovesciles structures can be obtained from nanotubes by altering the peptide concentration. ​8

          Nanowires:

          Similarly, Ihee and co-workers realized that the nanowires structures can be obtained from nanotubes by manipulating the free water content of the solution. 3

          Nanofibres:

          Bingbing sun and coworkers’ demonstrated self assembly of ultralong aligned dipeptide single crystals. 9

          As Oriented arrangement of single crystals plays a key role in improving the performance of their functional devices the group describes a method for the exceptionally fast fabrication (mm/min) of ultralong aligned dipeptide single crystals (several centimeters). It combines an induced nucleation step with a continuous withdrawal of substrate, leading to specific evaporation/composition conditions at a three-phase contact line, which makes the growth process controllable. These aligned dipeptide fibers possess a uniform cross section with active optical waveguiding properties that can be used as waveguiding materials. The approach provides guidance for the controlled arrangement of organic single crystals, a family of materials with considerable potential applications in large-scale functional devices.

          To initiate crystallization scratch is done on the silicon wafer in order to overcome nucleation barrier. Acidic or basic solvent system like increases solubility of FF dipeptides due to common ion effect. Structure of growing fibers like separation, thickness, and orientation can be controlled by concentration of , temperature and withdrawal speed.

          Microrods:

          Qi Li and co workers experimentally showed that duration of ultrasonification, concentration of FF molecules and solvent environment play significant role in determining morphology of the structures. ​ 8

          Microtubes:

          To obtain FF cationic dipeptide microtubes similar procedure to that of microrods is followed but the ethanol and water ratio must be 1:1 with 100μL of ethanol and water. Thus, FF cationic dipeptide microtubes can be obtained.​10

          Photo-luminescent properties of FF dipeptides:

          The delocalized p- electrons of aromatic systems and free proton transfer of hydrogen bonds during self assembly indicate that peptide supra-molecular semiconductors can transmit photons by continuous emission along the axis under excitation, allowing them to serve as optical waveguide and the emission can be modified to other wavelengths by incorporating various dyes implying a potential use of short peptide self assemblies in applications such as light harvesting, sensing and energy transfer.​3

          Introduction to Waveguides:

          A waveguide is a structure that guides waves, such as electromagnetic waves or sound, with minimal loss of energy by restricting expansion to one dimension or two. There are many types of waveguides for each type of waves. An optical waveguide is a physical structure that guides electromagnetic waves in the optical spectrum. Common types of optical waveguides include optical fiber and rectangular waveguides.

          Basic understanding of optical waveguides:

          The most used optical wave guide is step index fiber. It consists of a central dielectric core cladded by a materiel of slightly lower refractive index. The corresponding refractive index distribution (in the transverse direction) is given by

          n=n1;a>rn=n_1;a>r

           n=n2;r>an=n_2;r>a

          Where n1n_1 and n2n_2 (< n1n_1) is the refractive index of the core and the cladding respectively and ’a’ is the radius of the core.  

          Picture7.png
            Schematic of optical fibre showing core, cladding and its coating.

            Theory of operation of optical fibre:

            The guidance of light beam inside an optical fibre is only due to total internal reflection.

            An interesting effect known as total internal reflection can occur when light attempts to move from a medium having a given refractive index to a medium having a lower refractive index.

            Picture8.png
              Schematic showing total internal reflection of light that happens because of lower refractive index of cladding than core.

              When light is incident on the interface of two transparent medium, some of light is reflected and some is transmitted into second medium and the refraction should follow the Snell’s law. Now from Snell's law we have,

              According to Snell's law,  sinθ1sinθ2=n2n1{\frac {\sin \theta _{1}}{\sin \theta _{2}}}={\frac {n_{2}}{n_{1}}}

              Where n1n_1 and n2n_2 are the refractive indices of the two medium, θ1\theta_1is the incident angle and θ2\theta_2 is the angle of refraction.

              Picture9.png
                (a) For a light ray propagating from rarer medium to denser medium. (b) For a light ray propagating from denser medium to rarer medium.

                Now consider the two situation, firstly, when light is propagating from rarer to the denser medium and in this situation n1&lt;&ThickSpace;n2n_1&lt;\;n_2. So from Snell's law, we get and as a result the refracted ray is directed towards the normal.

                Secondly, when light is incident on denser medium n2&lt;&ThickSpace;n1n_2&lt;\;n_1 then the ray is bending away from the normal as. As we see in this situation if the incident angle θ1\theta_1 increases then also the refracted angle θ2\theta_2increases. Now for particular angle of incident, the reflected ray has an angle of 90090^0with the normal, then this particular angle is called critical angle θc\theta_c

                Picture10.png
                  (a) For a light ray at critical angle. (b) For a light ray at an angle greater than critical angle undergoing total internal reflection.

                  Formula for critical angle ( θc\theta_c) : We have

                  n1sinθ1=n2sinθ2n_1 sin\theta_1=n_2 sin\theta_2

                  At, θ1=θc,θ2=0\theta_1=\theta_c,\theta_2=0 So then,

                  n1sinθc=n2n_1sin\theta_c=n_2

                  This implies, θc=sin1n2n1\theta_c=sin^{-1}\frac{n_2}{n_1}

                  When the angle of incidence exceeds the critical angle (i.e., when θ1&gt;θc\theta_1&gt;\theta_c) there is no refracted ray and then the phenomenon is called total internal reflection (TIR).

                  Acceptance Angle and Numerical Aperture

                  Let us consider a step index optical fiber which has core and cladding having refractive index n1n_1 and n2n_2 respectively. Also n0n_0 is the refractive index of the outside medium of the fiber. Now a ray is incident on the entrance aperture of the fiber at an angle with the axis. If θ2\theta_2 be the angle of refraction then from Snell's law we get

                  n0sinθ1=n2sinθ2n_0 sin\theta_1=n_2 sin\theta_2

                  So, in order to keep the light inside the core the angle of incidence θ3\theta_3 at the core-cladding interface must be greater than the critical angle θc\theta_c  

                  Picture11.png
                    Demonstration of light ray passing through optical fibre.

                    From figure we can write,

                    n1sinθc=n2sin900n_1 sin\theta_c=n_2sin 90^0

                    θc=sin1n2n1 and θ3=900θ2\theta_c=sin^{-1}\frac{n_2}{n_1}  and  \theta_3=90^0-\theta_2

                    If θ1\theta_1 is increased, θ2\theta_2 increases and hence θ3\theta_3 decreases from the above equation. So there is a maximum value of for which is not less than and the ray undergoes total internal reflection at the core-cladding interface. This angle is called the Acceptance angle. θA\theta_A

                    Thus the Acceptance angle θA\theta_A is the maximum incident angle for which any ray is totally internally reflected at the core-cladding interface and therefore transmitted without loss. A cone of light of semi-angle is known as Acceptance cone.

                    Also this angle is a measure of the light gathering power of the fiber and called the Numerical Aperture (NA) of the fiber.

                    So , NA = SinθASin\theta_A =n21n22\sqrt[{}]{{n^2}_1-{n^2}_2}

                    Basic waveguide theory and concept of modes:

                    It is very important to know propagation characteristics of light in a waveguide. One has to solve Maxwell’s equations to determine the modes of waveguide. Transverse electromagnetic (TEM) modes are those in which neither electric nor magnetic field in the direction of propagation.

                    Transverse electric (TE) modes are those in which there is no electric field in the direction of propagation. These are sometimes called H modes because there is only a magnetic field along the direction of propagation (H is the conventional symbol for magnetic field).

                    Transverse magnetic (TM) modes are those in which no magnetic field is in the direction of propagation. These are sometimes called E modes because there is only an electric field along the direction of propagation.

                    Physical Understanding of the Modes:

                    The concept of modes, or Eigen-functions, is fundamental for all wave phenomena in physics like optics, acoustics and quantum mechanics. In optics and photonics, the concept of modes is well suited to describe emission and absorption, coherence and interference, propagation and dispersion. The concept of modes consists of two aspects: first, the modes are solutions for the propagation of the light; second, the number of photons in the different modes describes the transport of energy or information. The modes are basically defined by the properties of coherence and orthogonality, modes are orthogonal solutions of the wave equation, they do not interfere (the energy or optical power of a linear superposition of modes is equal to the sum the energy or the optical power of the individual modes).

                    It is a very necessary to understand physically about modes. We can say that, if a fiber has higher order modes then theoretically we can find out each angle of propagation for each mode.

                    Modes in Fibers and Coupling Efficiency:

                    In the following it will be shown how the basic definition of modes can be used to estimate number of accepted modes in a fiber, without being obliged to known the shape of the modes. The same approach is also used to estimate an upper limit for the coupling efficiency of different light sources into fibers and between different types of fibers.

                    Picture12.png
                      Schematic showing a light ray passing through step-index fibre.

                      A typical step-index fiber, as shown in Fig 10, is characterized by the numerical aperture,

                      NA = SinθASin\theta_A =n21n22\sqrt[{}]{{n^2}_1-{n^2}_2}

                      The corresponding solid angle of acceptance is given by,

                      Ω = π (NA)^2

                      and the area of acceptance (cross-section of the core)

                        AC=πa2A_C=\pi a^2

                      The number N of accepted modes can be estimated by comparing the volume in "phase-space" ΩmAm\Omega_mA_m required by a (spatial) mode, and the volume offered by the fiber ΩcAc\Omega_cA_c

                      The result is N = ωcAcωmAm \frac{{\omega_cA_c}}{{\omega_mA_m}}  .

                      For single-mode fibers N approximately equal to 1 and for multi-mode fibers N >> 1. The estimation from the above equation holds also for graded index fibers or any other type of waveguide. However, to get the accurate number of low order modes the exact shape and cutoff conditions of the modes have to be known. In the case of multi-mode transmission, the maximum information rate is limited by the spread of the group velocity of the different modes, the modal dispersion.

                      METHODOLOGY

                      Experiments

                      Coupling of light into multi mode and single mode fiber:

                      In order to gain better understanding of coupling and waveguiding phenomenon, we tried coupling laser beam into single mode and multi mode fiber using laser of operating wavelength 671 nm.

                      In our experiment we used SM600 thorlab optical fiber and GIF625 graded index multimode thorlab optical fiber.

                      Fiber_Coupling_Setup.jpg
                        Schematic of the experimental setup used for coupling of light into single mode and multi mode fiber

                        Characterization of Fiber used in our experiment

                        Multi mode fiber: GIF625 graded index multimode fiber

                        gif.jpg
                          Schematic of a typical multi mode fibre and its refractive index profile.

                          Core diameter: 62.5 ± 2.5μm

                          Cladding diameter: 125 ± 1μm

                          Operating wavelength range of the fiber: 800-1600 nm

                          Operating wavelength 671 nm

                          Numerical aperture: 0.275±0.015

                          Results:

                          Efficiency of multi mode fiber
                          Trail no Input power Output power Efficiency
                          1. 20 mW 17.2 mW 86%
                          2. 20 mW 18 mW 90%
                          3. 40 mW 34.6mW 86.5%
                          4. 40 mW 35 mW 87.5%
                          5. 100 mW 89.5 mW 89.5%

                            The coupling efficiency of our multimode fiber was found out to be 86%-90% by measuring the input and output power of the laser beam.

                          Single mode fiber: SM600 thorlab optical fiber

                          Operating wavelength range of the fiber: 633-780 nm

                          Operating wavelength: 671nm

                          Mode field diameter: 3.6-5.3 μm at 633nm

                          Cladding 125± 1μm

                          Coating: 245±15μm

                          Cut-off wavelength: 500- 600 nm

                          Numerical Aperture: 0.10

                          Core index: 1.46147

                          Cladding index: 1.45804

                          Results:

                          The coupling efficiency was found out to be 45%-48% by measuring the input and output power of the laser beam.

                          Methods

                          Description of the set up used:

                          Thermo-Optical Tweezers: The patterning of trails is performed on a glass cover slip where dispersion of the material to be patterned is exposed to a thermo-optical tweezers. It consists of a laser beam focused using a high numerical aperture objective lens. The thermo-optical tweezers is constructed around an inverted microscope. A 100×, 1.4 N.A. oil immersion microscope objectives is used to couple 1064 nm laser light. The laser power can be varied from 0 to 100 mW. The dispersion is placed on a coverslip. The surface of the coverslip is carefully cleaned with methanol and dried before conducting the experiment so as to eliminate the possibility of the presence of unwanted material. About 75 μL of solution is inserted into the holder, which is then placed in the microscope scanning stage which is operated using a joystick..4

                          setup.png
                            Schematic of the experimental setup for formation of trails

                            1. Description of the process of formation of SOMs trails:

                            The dispersion of Soft Oxometalates (SOMs) is taken on a coverslip. Some SOMs particles in the dispersion get adsorbed on the glass coverslip. When laser beam of wavelength 1064nm is tightly focused on these adsorbed particles, hotspots are formed due to high absorptivity of SOMs at this wavelength. Within few microseconds of the creation of hotspot, water vapour bubbles are formed in the vicinity of the hotspot. The difference in temperature between top and bottom surface of the water bubble leads to difference in their surface tension which eventually leads to Gibbs Marangoni flow of SOMs particles.

                            Now, the laser spot is moved using sample stage then the hot spot associated it follows the laser spot naturally. Due to the movement of hotspot, the bubble follows it due to Gibbs Marangoni convection causing deposition of the material at the base of the bubble.

                            trails.png
                              Microscopic images of linear pattern of SOMs

                              As the bubble is translated using the micrometer stage, continuous self assembly and subsequent crystallization of SOM particles occur in continuous rings, which finally forms the desired pattern whose width corresponds to the diameter of the rings.

                              Formation of FF microrings:

                              By following the procedure described above for the formation of SOM trials, peptide microrings are formed. A coverslip which has pre-existing SOM trails is taken and FF solution of concentration 10−2 M is drop-casted on the coverslip near the trails.The laser of wavelength 1064 nm is focused on such a trail after it is located using the camera fixed to the microscope. As stated earlier, the SOMs have high absorptivity at 1064 nm, and thus, a hot spot is created locally, which nucleates a bubble having a temperature gradient between the top and bottom surfaces of the bubble. The resultant surface tension gradient leads to Gibbs Marangoni flows that cause FF peptide particles to accumulate at the base of the bubble giving rise to FF microring structures.

                              ff rings..png
                                Microscopic images of FF microrings

                                The size of the ring can be tuned by changing the laser power and the time of laser exposure. We found that the micro ring, when locally excited at a spot by a focused 671 nm laser light, leads to circulation of light around it, with some losses appearing as light-leaks which are clearly observed on the surface of the micro ring.

                                FF WG.png
                                  FF microrings serving as waveguides when excited by wavelength of 671nm.

                                   Formation of CONGO RED (CR) dye doped FF microrings:

                                  Congo red Dye:

                                  Staining with Congo Red (CR) is a qualitative method used for the identification of amyloid in vitro and in tissue sections. CR is a direct diazo dye that is intended primarily for the colouration of paper products. 11 It is toxic and possibly carcinogenic and mutagenic. CR is the sodium salt benzidinediazo-bis-1-naphtylamine-4-sulphonic acid (formula: C32H22N6Na2O6N2C_{32}H_{22}N_6Na_2O_6N_2molecular weight: 696.66 g/mol;The Colour Index Number of CR is 22120.The CR staining technique has become a reliable and effective way to demonstrate amyloid deposits in tissues, and it is used for both research and diagnostic purposes. 12

                                  Researchers studying amyloidosis (disorders of diverse origin in which deposits of amyloid proteins are found) will routinely use this staining technique to detect the presence of amyloid in their tissues of interest.​13​Even after all these decades, CR still remains the gold standard test used by diagnostic pathologists to identify amyloid in tissues of patients with these conditions- perhaps the most widely known of which is Alzheimer’s Disease. As our main motto of this project is to find early and cost effective tool for detection of Alzheimer’s disease, we investigated the spectral responses of both FF microrings and Congo red dye doped FF microrings.​12

                                  crd ring.png
                                    Dark field microscopic image of CR doped FF micro rings

                                    We obtained CR dye doped FF microrings by drop casting FF solution which was doped with CR dye following the same procedure that is described for obtaining FF microrings.

                                    Dark field spectroscopy system:

                                    In our project, the spectral information of FF microrings and CR dye doped microrings were obtained by collecting the scattered light from the sample using dark field spectroscopy system merged with an inverted microscope. The spectral responses of CR doped FF microrings and FF microrings were subjected to comparative study using IGOR and MATLAB softwares.

                                    In our project of investigation of spectral responses of FF dipeptide microrings and CR doped FF microrings; we observe a special resonance in optics called the ‘Fano Resonance’ which occurs when a discrete localized state couples with continuum of states. We observe Fano resonance in these microstructures because these structures support both wave-guided modes and broadband modes under white light illumination. The coupling between these narrow and broadband modes gives rise to Fano Resonance.

                                    Fano Resonance:

                                    Fano resonance is a type of resonant scattering phenomenon that gives rise to an asymmetric line-shape which is produced due tointerference between a background and a resonant scattering process.​14​ In our project, asymmetric line shape is due to coupling of waveguide mode and the broadband mode from the source. Fano resonance possess an inherent sensitivity to changes in geometry or local environment: small perturbations can induce dramatic resonance or line shape shifts. ​15

                                    Theory of Fano Resonance:

                                    The scattered intensity due to interference between waveguide mode and broadband mode is given by,

                                    I(ω)=[(q+ω)21+ω2η+(1η)]B2I(\omega)=[\frac{(q+\omega)^2}{1+\omega^2}\eta+(1-\eta)]B^2

                                    Where, q is called the Fano asymmetry parameter that describes relative transition strength between discrete mode and continuum mode.

                                    Here Ω=Ω(ω)=ωω0γ2\Omega=\Omega(\omega)=\frac{\omega-\omega_0}{\frac{\gamma}{2}}

                                    Where ω0\omega_0and 𝜸 are the central frequency and the width of the narrow resonance respectively.

                                    Quality factor (Q) in Fano-resonances:

                                    Q-factor in fano resonance is defined as a dimensionless parameter which characterizs the strength of damping on the resonators.

                                    Q = Q=ω0Δω=ω0γQ=\frac{\omega_0}{\Delta\omega}=\frac{\omega_0}{\gamma}

                                    is the ω0\omega_0central frequency and Δω is the resonance width and is the damping.

                                    RESULTS AND DISCUSSION

                                    Fano resonance in FF microrings

                                    In order to study the spectral responses of FF microrings, they were subjected to dark field spectroscopy and the data was recorded. The spectral data of the FF microring is as shown in the Figure . It is clear from the figure that FF microring has three characteristic peaks corresponding to the wavelengths: first peak (for λ = 620-643 nm), second peak (for λ = 586-615nm), third peak (for λ = 565-573 nm).Using IGOR software we fit these peaks with fano formula to obtain corresponding fano parameters. Since CR dye has prominent response corresponding to the wavelengths of third peak, fitting this peak with the fano formula gives the corresponding fano parameters in which we are interested. 

                                    without dye ring43.png
                                      Theoretical fit of the third peak of the spectra obtained for undoped FF micoring with Fano formula

                                      The fano pararmeters corresponding to the third peak of FF micoring spectra is given in the table below:

                                       Fano parameters of undoped FF microring
                                      Fano parametersFF Microring
                                      q (Asymmetric parameter)-1.77±0.14
                                      (Energy corresponding to peak frequency ) 2.173±0.02
                                      γ (Damping factor)0.016±0.008
                                      Q (Quality factor)140±4.5

                                      Fano resonance in CR doped FF microrings

                                      As stated earlier CR dye is prominently used for detection of Alzheimers’ disease. In order to provide a tool for early detection of the Alzheimers’ we studied spectral responses of CR doped FF microrings.

                                      with dye ring43.png
                                        Theoretical fit of the third peak of the spectra obtained for CR doped FF micoring with Fano formula

                                        Similar to the case of undoped FF microrings, we obatined fano parameters corresponding to the third peak of the CR doped microring spectra. The fano parameters for CR doped FF microring is given below:

                                        Comparison of Fano parameters of FF microrings and CR doped FF microrings

                                        On comparing the fano parameters obtained from the spectral responses of FF microrings and CR doped FF microrings, we observe that there significant decrease of quality factor from 140.1 to 34.56 due to increase in damping factor. Significant change in the quality factor assures that these FF microrings may serve as effective tool in detection of Alzheimer’s Disease.

                                        CONCLUSION

                                        In conclusion, self assembly of nano and microstructures plays extremely important role in pioneering nanoscience and nanotechnology to a new dimension and achieving wide range applications in the field of electronics, medicine, material science etc. Diphenylalanine (FF) being one of the simplest bio molecular building block possessing various physical and chemical properties including waveguiding property of our interest. Various studies have demonstrated procedures and techniques to obtain different morphologies of FF structures. However, all these methods lack control in spatial location of the microstructures. In this project we make use of a novel technique called ‘Laser Induced MicroBubble Technique (LIMBT)’ using thermo optical tweeezers setup to obtain FF microrings. Using the same technique we obtain CR doped FF microrings and observe waveguiding popeties of these microstructures so obtained. To investigate the spectral responses of undoped and doped FF microings we subject them to dark field spectroscopy and notice Fano resonances occurring due to coupling of continuum mode from the source and the waveguide mode. In our project, we observe significant change in the quality factors of the fano parameters of FF microrings and CR doped FF microrings. This shows that these FF microstructures are suitable for detecting Alzheimer’s disease.

                                        REFERENCES

                                        Congo Red – A Special Stain For Alzheimer’s Disease. Bitesizebio. Retried from:​https://bitesizebio.com/13435/were-all-doing-the-conga-actually-no-its-the-congo-red-histology-stain/

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                                        ACKNOWLEDGEMENTS

                                        I would like to thank the Indian Academy of Sciences (IASc –INSA) for providing me a great opportunity to work at an esteemed institute.I am very fortunate to get this opportunity from Indian Academy of Sciences Bangalore.This internship was a very unique experience that helped me delve into uncharted fields and opened me up to some amazing fields of work.

                                        I owe my profound gratitude to my guide Dr. Ayan Banerjee, Department of physical sciences, Indian Institute for Science Education and Research Kolkata for his most valuable guidance and support.

                                        I extend my sincere gratitude to Mr.Roshan Tiwari who took keen interest and guided me throughout the project by roviding all necessary information.

                                        I would also like to thank Mr. Souvik Sil and Mr. Subhrokoli Ghosh for discussing the topics regarding my project and clearing my doubts which enhanced my understanding of the subject. I also thank all the lab members of our lab for their constant support and guidance.

                                        Last but not the least I would like to thank my parents and family for their moral support and my friends who boost me up every time.

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