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Nanosystems Engineering on Microfluidic Platform: Potential Applications in Healthcare and Energy Domain Nripen Chanda Sr. Scientist Council of Scientific & Industrial Research Organization CMERI, Durgapur 713209, India CMERI: MST Labs The Microsystems Technology Laboratory was established in the year 2006 at The Central Mechanical Engineering Research Institute, Durgapur, West Bengal, India 2 Research focus • Micro-Nano Scale Process Technologies • Micro-Nano Systems and Devices • • • • Lab-on-chip (microfluidics) Nanosensors (microfluidics): DBT funded Self cleaning patterned surfaces Textured solar cell: (AGATHA, IndoEU-FP7) • Geometry: 100nm – 500µm – Micro machining (material removal) – Micro molding, laser sintering – Electro-deposition – Chemical approach, self assembly/organization 3 Theme of presentation Integration of nano-scale materials into new and traditional micro/macro devices for biomedical and energy applications Nano materials Micro devices Energy & Bio-applications 4 Nanosystems engineering Nanosystems engineering deals with the design, development and characterization of materials (sized between 1 to 100 nanometer in at least one dimension). Water molecule Nanospheres Nanotubes Nanoshells Human cell A period Tennis ball 5 Features of nanosystems engineering Interdisciplinarity Mechanics, electronics, fluidics, biology, and chemistry Heterogeneity Semiconductor, ceramic, glass, organic Multifunctionality Sensing & drug delivery, memory storage and energy applications, Integration Integration of nanotechnologies and smart systems into new and traditional materials, e.g. textiles, glass, paper 6 Design of nanoscale materials Functional Molecules 1. Peptide Markers 2. Antibody Analytes of interest 3. Protein 4. Nucleic acid Nanoparticle core as carrier 5. Drug 6. Fluorescent marker 7. Charged species Multifunctional Nanomaterials • sense, diagnose, describe and qualify a given situation; • suggest or implement appropriate actions; • interact with the user, the environment or another smart systems. 7 Looks of nanoscale materials Spheres Rods Cages Gold nanoparticles • • • • Nano Letters, 2009, 9, 1798 Anal. Chem., 2012, 84(21), 9478-84 Proc. Natl. Acad. Sci. (PNAS) USA, 2012, 109, 12426 Bioconjugate Chem., 2014, 25, 1565 8 Properties of nanoscale materials • Lateral dimension: 1 – 100 nm Polymeric nanomaterials • Exhibit novel physical, electronic and optical properties due to – Two dimensional quantum confinement – High surface to volume ratio • Potential applications in wide range of devices – – – – Sensors and actuators Photovoltaic devices – solar cells Fuel cells, energy storage Imaging agents, drug delivery systems Micro System Technology, CSIR-CMERI, Durgapur, India 9 Applications of nanoscale materials Point-of-Care diagnostics − Small and highly sensitive sensors for biologically relevant molecules/ions detection − Biochemical analysis (protein/cell analysis) Toxin detection device In-vitro drug delivery applications − Drug screening − Improved delivery of poorly water soluble drugs − Imaging of drug delivery sites using imaging modalities Targeted drug delivery 10 Applications of nanoscale materials Fuel cell applications − Fuel cells use hydrogen and air as fuels and produce water as by product − The technology uses a nanomaterial to produce electricity Schematic of a fuel cell – Micro System Technology, CSIR-CMERI, Durgapur, India 11 Use of nano-materials in cancer cell detection Biological differences between normal and abnormal endothelium cells for cancer tissue Normal tissue Cancer tissue ♦ Tumor endothelium is structurally irregular, heterogeneous, and leaky (larger endothelial pores) compared to normal vessels. ♦ Through circulatory system, nanoparticles enter into tumor tissue through endothelial pores (100 nm to 2 µm), known as Enhanced Retention and Permeability effect (passive targeting). ♦ Tumor vessels have been shown to exhibit receptors (e.g., GRP, EGF) that can serve as potential targets for targeted nanoparticles (active targeting). 12 Tumor Avid Gold Nanoparticle (GNP) Starch-GNP • GNP • Carbohydrate Lung GumArabic- GNP Protein-GNP Liver Prostate ● GNP ● Glycoprotein • GNP • Peptide/Antibody Gold nanoconjugate Biomolecule Target GNP-starch, GNP-dextran Carbohydrate Lungs, Liver cancers GNP-gumArabic, GNP-gelatin Glycoprotein Liver cancers GNP-Polyethylene glycol Polymer Spleen abnormality GNP-GRPR Peptide Prostate, breast cancer GNP-EGFR Antibody, Peptide Pancreatic and colon cancer 13 Tumor Homing Small Biomolecule Advantages in “Small Bio-molecule” delivery platform : (a) ease in penetration of tumor vascular endothelium, (b) increased diffusion rate in tissue, (c) rapid blood clearance, and (d) low immunogenicity Thioctic acid modified GRPR peptide (SS-BBN) for prostate and breast cancers Thioctic acid modified EGFR peptide (SS-EGF) for pancreatic and colon cancers 14 Interaction with Cancer Cell (In-vitro) GNPs internalized into cancer cells by endocytic mechanisms by using PEPTIDE – RECEPTOR interaction. Nuclear envelop Mitochondria GNP in cytoplasm Cell membrane TEM image of PC-3 cells after treatment with GNP-BBN Chanda N et. al. Nano Letters, 2009 & Bioconjugate chemistry, 2014 The corresponding XEDS spectrum 10 µm Dark field images of PC-3 cells after treatment with bombesin conjugated GNP (GNP-BBN) 15 Cancer Tissue Imaging (In-vitro) Colon tissue with and without GNP-EGFR peptide, viewed under dark field microscopy No back scattered light observed Scattered light observed due to attached GNP In DF imaging, single gold nanoparticle can be imaged at very low concentration of GNP-EGFR peptide treatment. Significant contrast of peptide conjugated gold nanoparticles provides new opportunities for targeted delivery and molecular imaging. 16 Detection of Circulating Tumour Cells Photoacoustic imaging has potential for the early diagnosis of CTC to prevent metastasis in humans. Schematic illustration of photoacoustic imaging CTCs emerge from the primary site into blood vessel and spread to other organs Detection of photoacoustic signals by irradiating AuNPs tagged 15 prostate cancer cells/100µL under flow show strong photoacoustic signals (Right) 17 Chanda N et. al. Pharmaceutical Research, 2011, 28,279–291. Use of microfluidic platform • Sample savings – smaller piping needs smaller volumes of fluids • Faster analyses – surface area-to-volume (SAV) ratio increases and thus renders any surface phenomenon to become more dominant than the volume factor – diffusion, surface tension, and surface effects dominate – This can actually lead to faster reactions! • Integration – combine lots of steps onto a single device 18 Microfluidics concept Microfluidics deals with the science and technology of the fluid behavior on the fundamental microscopic level to give rise to very powerful techniques in controlling and measuring chemical reactions and physical and biological processes on the micro and nanoscale. Laminar vs Turbulent Flow Re = ρUdh/ μ - μ is kinematic viscosity of the fluid U is a characteristic velocity of fluid dH is hydraulic diameter of channel. 19 Pressure driven flow and particle migration • Shear induced inertial lift force Parabolic velocity profile of Poiseuille flows Particles roll down towards microchannel walls Directed away from microchannel center • Saffman Fl =1.615 ρ ν 12 d 2 u f -u p f 12 du dy duf sgn dy - uf is the fluid velocity at the location of mass centre of the particle, - up is the particle velocity, - (duf/dy) is the shear rate, - d is the particle diameter and - ρ and µ are the fluid density and viscosity. 20 Pressure driven flow and particle migration • Wall induced lift force (FWL) Flow field around particles disturbed due to presence of walls Wall induced asymmetric wake exerts a lift force on particles Directed away from the microchannel wall • Segre and Silberberg (1962) Particles equilibrate around channel periphery “Tubular pinch effect” Segre and Silberberg, J. Fluid Mech., 1962 Fl > FWl FWl > Fl FWl ~ Fl Fl FWl Equilibrium position achieved Nanoparticles focusing in microchannel Carrier phase (H) = 0.15 mm, (L) = 25 mm,(W) = 3.3/0.8 mm Converging zone Nanoparticles laden fluid Carrier phase Diverging zone Accumulation of fluorescent nanoparticles (500 nm) at fluid-fluid interface Higher concentrations of nanoparticles at interfaces Nanoparticles distribution: fluorescence intensity vs. distance along the width of channel. Arun RK, Chaudhury K, Biswas G, Chanda N*,Chakraborty S*. Lab on a Chip, 2014, 14, 3800 22 Nanoparticles focusing in microchannel Hydrodynamic focusing of fluorescent nanoparticles (30 nm) in microchannel. (a-d) Microscopic fluorescent images at 470 nm wavelength at different flow ratio (25:25, 25:50, 25:100, 25:250). (e-h) Plots of fluorescence intensity vs. distance along the width of channel. The maximum concentration of nanoparticles was observed at two interfacial zones with separation width of 100 µm at flow ratio of 25:50. 23 Nanoparticles focusing in microchannel Absence of nanoparticles distribution 200 μm (b) (a) 100 200 μm 100 (e) (c) (g) (h) 90 Intensity Intensity Intensity 50 (d) 105 100 (f) 80 75 200 μm 60 80 Intensity 200 μm 60 40 75 60 25 20 0 0 175 350 525 Transverse distance (m) 700 0 150 300 450 Transverse distance (m) 600 40 0 150 300 450 Transverse distance (m) 600 45 0 150 300 450 Transverse distance (m) Hydrodynamic focusing of fluorescent nanoparticles (30 nm) in straight microchannel. 24 600 Underlying physics leading to particle focusing duP/dt = fD + fG + fM + fS dxP/dt = uP Distribution of the streamlines on an x–y plane passing through the central axis of a converging–diverging channel 3D simulation results of the distributions of 45 nm particles in a converging diverging channel at a flow rate of 25 μL min−1, for both the carrier and the dispersed phases. The flow was imposed along the x direction and the particles were initially distributed axially around the central axis of the channel. 25 Controlled reaction in microchannel AgNO3 + NaBH4 SDS AgNP (40 nm) Superimposed microscopic image demonstrating the interaction of AgNPs and H2O2 throughout the patterned micro-channel AgNP + H2O2 Ag2O + H2O NH4OH [Ag(NH3)2]+ 26 Controlled reaction in microchannel SEM image of the Ag ion precipitate formed at the interfaces at different sheath flow rates (a) 25 µL/min, (b) 50 µL/min, (c) 100 µL/min and (d) 250 µL/min for 30 min. (a) Magnified SEM image of silver ion precipitate which appears as flower-like structure. (b) EDS spectrum of Ag ion precipitates showing the presence of Ag. The peaks of the Si and O elements are due to the Si substrate. 27 Gold nanosensor in Lab on a chip: As detection PDMS Channel DI Water with Arsenic ions θ = 45° Gold nanosensors AuNS M As3+ M M M M M M M M M M M M Nanosensor M M M M M M M M M M M M M M M M M M M M M M M M M M M M Agglomerated nanosensors Binding of As3+ ions to AuNS to form BLUE aggregates 28 Flow in paper channel Dye flow in Y-shaped paper channel (Flow rate: 2-3 µl/min) Paper contains microchannels made of an embedded network of fibers through which a liquid can pass automatically due to capillary flow. This self-pumping property of the paper substrate along with its low cost and easy availability makes it an attractive substrate for the present studies. SEM picture of filter paper 29 Paper based microfluidics for arsenic detection Gold nanosensor (Au-TA-TG) in Petri dish - Arsenic recognition site Binds with arsenic Au-TA-TG Thioctic acid Thioguanine (spacer) (probe) As Paper channel Nanoparticle aggregation Arsenic sample in Petri dish Nath P, Arun RK,. Chanda N* “A paper based microfluidic device for the detection of arsenic using a gold nanosensor” RSC Adv., 2014, 4, 59558-59561 30 Sensitivity of gold nanosensor As AuNS Control 1 Absorbance 0.3 10ppm 2 0.2 1ppm 0.1ppm 0.01ppm 0.001ppm 3 4 λmax= 520 nm 1 5 Appearance of a new broad peak at 620 nm 2 0.1 3 4 6 1. 2. 3. 4. 5. 6. Au-TA-TG solution Au-TA-TG + 10ppm As3+ Au-TA-TG + 1ppm As3+ Au-TA-TG + 0.1ppm As3+ Au-TA-TG + 0.01ppm As3+ Au-TA-TG + 0.001ppm As3+ 5 6 0 300 400 500 600 Wavelength (nm) 700 800 31 Arsenic detection on paper microchannel 10ppm Gold nanosensor Arsenic solution 1ppm 0.1ppm 0.01ppm 0.001ppm Control Blue precipitate 15mm 3mm On paper strip 10ppm 1ppm 0.1ppm 0.01ppm 0.001ppm Control 32 Selectivity of gold nanosensor towards arsenic 2 Au-TA-TG + all ions (10ppm) including As (III) A620/A520 1.5 1 Au-TAT-G + 10ppm Ca(II) Au-TA-TG + 10ppm K(I) Ca(II) K(I) Au-TAT-G + 10ppm Mg(II) Au-TA-TG + 10ppm Na(I) Au-TA-TG + Au-TA-TG + all ions 10ppm Fe(II) (10ppm) except As (III) 0.5 0 As(III) Mg(II) Metal ions Na(I) Fe(III) mixture of ions except As(III) 33 Construction of detector module To power source White LED LCD Display Sample Color sensor Microcontroller Internally silver coated box RGB array Prototype of the heavy metal ion detector Photodetector 34 Uric acid detection on paper strip Paper strip (+)AuNPs TMB oxTMB H 2 O2 oxTMB Colourless (Bluish green) Uric acid Glass slide TMB (+)AuNPs (colourless) (Bluish green colour) Red TMB TMB + H2O2 H2 O2 (+)AuNPs Blue Oxidized TMB Uric acid Uric acid Colourless Capillary flow TMB 35 Paper strip assay for uric acid detection Increasing uric acid concentrations 0 ppm 33 ppm 50 ppm 83 ppm 100 ppm Kumar A, Hens A, Arun RK, Chatterjee M, Mahato K, Layek K, Chanda N* Analyst, 2015, DOI: 10.1039/c4an02333a 36 Drug delivery applications using Lab-on-a-chip technology Drug delivery means controlling mass transport at micro-scale. Drug formulation Drug delivered to target cells Drug taken by the cells Different stages of drug delivery 37 Drug delivery applications using Lab-on-a-chip technology HSA Microfluidic research paves the way to create a more in-vivo like cellular microenvironment in-vitro in which cellvolume-to-extracellular fluid volume is usually greater than one. HSA In-vitro microenvironment In-vivo blood capillaries PLGA-Rh-TG nanoparticles PLGA HSA Rhodamine + TG TEM 38 Polymeric NPs as drug delivery systems PLGA-Rhod-TG nanoparticles HSA Transport through microchannel No change in physicochemical properties of the nanoparticles 39 Dual-task paradigm: Therapy and Imaging Imaging capability Therapeutic response 40 Microfluidic fuel cell: Micro-power source What is hindering the success of fuel cells in the portable arena? PDMS channel with copper electrodes Formic acid O2 (Air) CO2 DAQ + Graphite anode Paper channel Tissue Paper Graphite cathode H2SO4 Glass plate Arun RK, Halder S, Chanda N*, Chakraborty S* Lab on a Chip, 2014, 14, 1661-1664 41 Paper based microfluidic fuel cell HCOOH 2HCOOH 2CO2 + 4H+ + 4e- Anode CO2 Tissue Paper Cathode O2 (Air) O2 + 4H+ + 4e- 2H2O H2SO4 10 mm Engraved channel parameters were as follows: length (l) = 25 mm, width (w) = 6 mm and height (h) = 0.1 mm. 42 Micro-fuel cell performance: OCP 2.0 1.5 t = 1000 minutes Fuel required = ~ 1 ml E (volt) t = 3500 minutes Fuel required = 2.5 ml 1.0 0.5 0.0 1000 2000 t (min) Systematic experimental plan Number of strokes (k) of Hb pencil Area of the graphite electrode Load variation of a single cell (2-10 ohm) Fuel cell in series Ag-NP particles on paper 3000 4000 Polarization and Power Curve Max. current density was 660 mA-cm-2 Max. power density 32 mW-cm-2 Effect of pencil strokes and its area as graphite electrodes 44 Effects of nanoparticles & series connection Effect of AgNPs : Potential increases, > 0.3 volt Six cells in series connection 45 Conclusions Design of nanoscale materials and study their transport properties in microfluidic domain for biomedical (healthcare, biochemical analysis) and environmental applications - Microfluidic platform to demonstrate the drug delivery efficacy - Toxin and protein detection - Biologically important molecules, such as amino acids, hormones, uric acids etc. detection Design and development of fluidic devices with applications in energy systems. 46 Current research projects 1. “Design and development of biocompatible micro/nano scale materials and study their colloidal properties in microfluidic domain” funded by CSIR India (OLP 101512). 2. “Design and Development of Gold-Iron Oxide Based Smart Magnetic Nanosensor for Detection and Separation of Heavy Metal Ions” funded by Department of Biotechnology, India (GAP 101612). 3. Nano patterning of metallic and polymeric thin films funded by CSIR India (ESC0112) Mr. Ravi K Arun Ms. Peuli Nath Ms. Puja Mitra Group members Ms. Preeti Singh Ms. Nivedita Priyadarshini Ms. Eleena Tom THANK YOU Ms. Manosree Chatterjee 47