Microparticle Based Assays

Transcription

1 Microparticle Based Assays Last Class: 1. Mass Transport : Advection Diffusion Equation 2. Boundary Phenomena 3. Physical Properties as a Function of Concentration 4. Mixing/Separation/Purification of Bioparticles 5. Cellular Microfluidics Today s Contents: 1. Microparticles as Biological Tools 2. Surfactant and Micelles 3. Chemical Modifications of Surfaces 4. Experimental Methods of Characterization 5. Biochemical Characterization 6. Molecular Manipulation 1

2 Why Microparticle Based Assays? Two categories are usually interested in the study: The first category is the particles of biological interest that are naturally present in the biological systems and on which it is necessary to obtain some information. The second category deals with artificial particles that are manufactured by chemical synthesis or by genetic modification, as tools to perform a function in the process (observation, characterization, or manipulation). Consideration: High Sensitivity, High Precision (Single Cell), High Efficiency, Less Waste. 2

3 Some Biological Targets Biopolymers DNA, RNA Proteins Micro/Nano Organisms Eukaryotic Cells Bacteria/Viruses 3

4 Microparticles as Biological Tools Synthetic microparticles find a natural use either as a macroscopic handle for the manipulation of molecules or cells, or to add or to enhance a signal in the various detection schemes. This last category includes immunofluorescence or immunoelectron microscopy in which a fluorescent dye or a metal colloid is coupled to an antibody that specifically targets the molecule of interest. 4

5 Fluorescent Particles/Molecules Fluorescence is one of the major tools used in biochemistry/ biotechnology. The high sensitivity of the technique and the numerous available coupling strategies make it a very versatile routine tool. Jablonski energy diagram Excitation (black) and emission (gray) fluorescence spectra of fluorescein. 5

6 Exogenous Fluorophores : Organic Molecules Fluorophores are characterized by their excitation and emission spectra; their quantum yield, defined as the number of emitted photon per absorbed photon, quantifies their efficiencies. However, they are often toxic and their use is consequently restricted mostly to fixed cells. Unfortunately, these molecules cannot switch indefinitely between their excited state and their ground state. After a certain number of these transitions, they permanently lose their fluorescence properties, a phenomenon called photobleaching that is enhanced by the presence of dissolved oxygen in the solution. 6

7 Exogenous Fluorophores : Micro/Nano Particles Fluorescent latex beads are plastic beads loaded with organic fluorophores. These particles are synthesized by emulsion polymerization in which the hydrophobic organic monomer is encapsulated by surfactant molecules in a micelle and then polymerized in the water phase. These relatively large particles are quite useful to reveal the structures of larger structures or to probe flows in microchannels geometries. Their surface can be tailored to match one s particular application by grafting particular molecules to them. 7

8 Endogenous Fluorophores : GFP Green fluorescent protein (GFP) is a naturally fluorescent protein present in the jellyfish Aequorea Victoria. The GFP reporter gene can be fused by genetic engineering to the one of the protein of interest so that the resulting protein is a fusion of both, coupling the desired function with fluorescence. This way, proteins in living cells can be directly observed by fluorescence. 8

9 Exogenous Fluorophores : Quantum Dots (QDs) Quantum dots (QDs) are fluorescent nanoparticles (typically 10 nm) whose use in biology oriented applications is relatively recent. These inorganic particles are made of semiconductors, and besides their small size, have a few remarkable properties: (1)They all share a broad excitation spectrum in the blue and their emission wavelength depends only on their size. (2)They are extremely bright and (3)show practically no photobleaching. (4)Moreover, they are small enough to be incorporated in many systems, even at the surface of live cells. 9

10 Other Particles : Gold Nanoparticles The main use of gold nanoparticles is electron microcopy. They can be coupled to antibodies by electrostatic nonspecific adsorption. When the target protein is present, the nanoparticles couple specifically to it and appear as distinctive tiny black spots in transmission electron microscopy. The diameter of gold nanoparticles determines the wavelengths of light absorbed. The colors in this diagram illustrate this effect. 10

11 Surfactant and Micelles Surfactants (also called amphiphiles) are molecules composed of two antagonistic parts: a hydrophilic polar head and a hydrophobic nonpolar tail. Phospholipids that are the constituents of the cellular membrane belong to this category. In fact, they tend to aggregate into micelles. In other words, below c* called the critical micellar concentration (CMC), surfactant molecules are individually solubilized; above this concentration, they tend to aggregate in micelles. 11

12 Surfactant and Micelles (Cont.) Micelles are not restricted to small surfactant molecules. In particular, some block copolymers can be tailored to make micelles particularly well suited to drug delivery applications. From geometric arguments, when the tail group of these molecules is too bulky for a good packing into micelles, bilayers can self assemble and lead to the formation of vesicles. This is for instance the case for most of 2 chains surfactants such as phospholipids that are the major constituents of cell membranes. 12

13 Colloidal Stability The scientists Derjaguin, Verwey, Landau and Overbeek developed a theory in the 1940s which dealt with the stability of colloidal systems. DVLO theory suggests that the stability of a particle in solution is dependent upon its total potential energy function V T. This theory recognizes that V T is the balance of several competing contributions: where A is the Hamaker constant and D is the particle separation. The repulsive potential VR is a far more complex function. where a is the particle radius, π is the solvent permeability, κ is a function of the ionic composition and ζ is the zeta potential. 13

14 Colloidal Stability (Cont.) To maintain the stability of the colloidal system, the repulsive forces must be dominant. There are two fundamental mechanisms that affect dispersion stability : Electrostatic or charge stabilization this is the effect on particle interaction due to the distribution of charged species in the system. 14

15 Colloidal Stability (Cont.) Steric repulsion this involves polymers added to the system adsorbing onto the particle surface and preventing the particle surfaces coming into close contact. If enough polymer adsorbs, the thickness of the coating is sufficient to keep particles separated by steric repulsions between the polymer layers, and at those separations the van der Waals forces are too weak to cause the particles to adhere. 15

16 Electric Double Layer (EDL) A net charge at the particle surface affects the distribution of ions in the surrounding interfacial region, resulting in an increased concentration of counter ions, ions of opposite charge to that of the particle, close to the surface. Origin of surface charge by ionisation of acidic groups to give a negatively charged surface Origin of surface charge by ionisation of basic groups to give a positively charged surface. 16

17 Zeta Potential The general dividing line between stable and unstable suspensions is generally taken at either +30 or 30 mv. Particles with zeta potentials more positive than +30 mv or more negative than 30 mv are normally considered stable v2/css/h1015v2_38.htm 17

18 Dispersing Agents : Anchor Groups Ionic or Acidic/Basic Groups Hydrogen Bonding Groups Polarizing Groups Solvent Insoluble Polymer Blocks Derivatives of the Dispersed Particle 18

19 Dispersing Agents : Polymeric Chains The molecular weight of the polymeric dispersants must be sufficient to provide polymer chains of optimum length to overcome Van der Waals forces of attraction between pigment particles. If the chains are too short, then they will not provide a sufficiently thick barrier to prevent flocculation. When the chains are too long, they have a tendency to "fold back" on to themselves 19

20 Dispersing Agents : Surfactants Surfactants are conventional low molecular weight dispersing agents. Surfactant molecules are able to modify the properties and, in particular, they lower the interfacial tension. The stabilization mechanism of surfactant like dispersing agents is electrostatics. 20

21 Chemical Modification of Surfaces : Non Specific Adsorption For these micronanoparticles that have a large surface/volume ratio, surface related problems and surface chemistry are of paramount importance. The first method that comes to mind to immobilize biomolecules on surfaces is to rely on nonspecific adsorption. Metal surfaces in particular gold and oxide surfaces in particular SiO 2 are good templates for chemical modifications. Hydrophilic/Hydrophobic Surface Charges 21

22 Chemical Modification of Surfaces : Non Specific Adsorption Surface defects Porous matrix Rough surface 22

23 Chemical Modification of Surfaces : Specific Binding ELISA 23

24 Experimental Methods of Characterization : Optical Principle of fluorescence microcopy. In this particular configuration, the excitation light and the fluorescence emission go trough the objective (epifluorescence). Resolution 24

25 Experimental Methods of Characterization : Optical Scanning Nipcow disk 25

26 Experimental Methods of Characterization : Optical Super resolution Microscopy 26

27 Experimental Methods of Characterization : Non Optical 27

28 Atomic Force Microscope (AFM) Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very highresolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever according to Hooke's law. Depending on the situation, forces that are measured in AFM include mechanical contact force, van der Waals forces, capillary forces, chemical bonding, electrostatic forces, etc. 28

29 Scanning Tunneling Microscope (STM) A scanning tunneling microscope (STM) is an instrument for imaging surfaces at the atomic level. For an STM, good resolution is considered to be 0.1 nm lateral resolution and 0.01 nm depth resolution. When a conducting tip is brought very near to the surface to be examined, a bias (voltage difference) applied between the two can allow electrons to tunnel through the vacuum between them. The resulting tunneling current is a function of tip position, applied voltage, and the local density of states (LDOS) of the sample. STM image of gold surface 29

30 Biochemical Characterization : SPR The surface plasmon resonance (SPR) technique is used to quantify the amount of material on a surface. In biotechnology, this technique is used to detect and measure in real time the kinetic parameters of the interaction between two or more molecules. One of the strengths of the technique is that it does not require labeling the molecules. The SPR effect is based on the interaction of light with a metallic surface in the conditions of total internal reflection which means that, in the absence of a metallic layer, the light propagating in the solid (a glass prism) is totally reflected. In these conditions, an evanescent wave exists at the surface of the glass. 30

31 Application of SPR A thin metallic layer present on the glass surface will not qualitatively modify this picture; most of the light is still totally reflected. However the evanescent wave can couple with the free electron clouds of the metal to create a plasmon (a cloud of excited electrons). When requirements are fulfilled, energy is effectively transferred to the plasmonsand thisresults in a minimum in the reflected intensity. 31

32 Molecular Manipulation : Force Measurement One of the main micromanipulation techniques at the molecular scale uses the AFM described earlier. With this instrument, forces between individual objects such as proteins can be measured. The principle is quite simple: one of the interacting proteins is immobilized on the tip of the instrument and the other one on a facing solid surface. The tip and the surface are first bought into contact and then separated. The force necessary to separate them is measured by the deflection of the cantilever. Where r f is the so called loading rate (product of the cantilever spring constant by the velocity of separation) and x β a characteristic length of the bond. 32

33 Molecular Manipulation : Optical Tweezers Optical tweezers (OTs) use a highly focused optical beam to trap particles at the focus. When a laser is focused through a high numerical aperture microscope objective, it defines a well defined light cage in which not only the intensity is maximal but where the gradients in light intensity are also extremely strong. Energy Trap 33

34 Molecular Manipulation : Optofluidics 34

35 Molecular Manipulation : Optoelectronic Tweezers (Chiou et al., Nature, 2005) 35

36 Molecular Manipulation : Flow Based Techniques Flows have also been used to align molecules: Elongated rigid objects such as microtubules orient naturally in the direction of the flow and a receding meniscus can be used to perfectly align DNA molecules in the direction of drying. This molecular combing is performed on a modified surface and in the right ph conditions in order to have one end of the DNA stick to the surface. 36

37 Molecular Manipulation : Flow Based Techniques DEP ACEO Flow λ phage DNA (48.5 kbp, Invitrogen ) labelled with 1 lm YOYO 1 fluorescent dye. 37

38 Molecular Manipulation : Fluidic Self Assembly (FSA) Fluidic self assembly using a template surface and employing liquid stream to select appropriate components, is another approach. The figure below illustrates the general procedure in fluidic self assembly. General principle of fluidic self assembly uses a template surface and employing a liquid stream. 38

39 Molecular Manipulation : Fluid Self Assembly (FSA) 39

40 Molecular Manipulation : Droplet Techniques Single cell studies require that virtually all drops contain at most one cell, so that the majority of drops contain no cell at all since the encapsulation process follows Poisson statistics. where n is the number of cells in the drop and λ is the average number of cells per drop; we adjust λ by controlling the cell density. 40

41 Molecular Manipulation : Droplet Techniques OEW 41

42 Applications : Sorting OEW+OET 42