Week 9 - lecture 8.pptx
- 1. BIOSENSORS BEN213 Nanobiosensors WEEK-9 SpringSemester Faculty: AsstProf.Dr.BetülGürünlü Faculty of Engineering & Natural Sciences Uskudar University
- 2. T opics that will be covered in the course History of biosensor development, applications and requirements of biosensors and classification Principles of molecular recognition and transduction signal acquisition Sourcesof Biological Recognition elements – enzymes/proteins, ssDNAs, antibody and Others Design considerations for use of recognition elements in biosensors Modeling of reactions for various biosensor applications‐ electrochemical, optical, piezoelectric, colorimetric, fluorometric and others. Modification of sensorsurfaces and immobilization techniques Covalent modification of surfaces using surface chemistry Self Assembled Monolayers (SAM) and adsorptions Other ways to immobilize biological macromolecules on various solid surfaces Detection methods and Physical Sensors Electrodes/transducers – electrochemical (amperometric, potentiometric, and conductimetric transductions) Other sensors ‐ for e.g., optical sensors (colorimetric/fluorimetric/luminometric sensors), Surface Plasmon Resonance(SPR) sensors,and piezoelectric resonators. Fabrication of biosensors Miniaturization‐application of nano‐materials, nanoparticles, carbon nanotubes (CNTs) and others Biocompatibility – stability, reproducibility and repeatability of biomolecules on transducer surfaces Data acquisition, statistical and error analysis Inter and Intra‐assays and Coefficient of variation (CV) Signal to noise ratio Normalization/optimization and signal retrieval Examples of commercial biosensors
- 4. NANOSENSORS: Exploring the Sanctuary of Individual LivingCell: The combination of nanotechnology, biology, advancedmaterials and photonicsopensthepossibility of detecting and manipulating atomsand moleculesusingnano-devices, which have the potential for a wide variety of medical usesat the cellular level. R ecently reported information showed the development of nano-biosensors and in situ intracellular measurementsof singlecellsusing antibody- based nanoprobes. The nano-scale size of thisnew class of sensors also allows for measurements inthe smallestof environments. Onesuchenvironment that has evokeda great deal of interestisthat of individual cells. Using these nanosensors,it is possible to probe individual chemicalspeciesand molecular signaling processesin specific locations within a cell. Studies have shownthat insertion of a nano-biosensor into a mammalian somatic cell not only appears to have no effect on the cell membrane, but also does not effect the cell's normal function. The possibilities to monitor in vivo processeswithin living cells could dramatically improveourunderstandingof cellular function, thereby revolutionizing cell biology. New‐generation nano‐engineered biosensors, enabling nanotechnologies and nanomaterials
- 5. Nanotechnology involves development of materials (and even complete systems) at the atomic, molecular, or macromolecular levels. The dimensional range of interest is approximately 1-500 nm. Inthefield of biosensors,carbon nanotubes,thetypical elementof nanotechnology, have received considerable attention because of their inert properties conducting behaviour and high-surface area. Particularly, their promotional ability for electron-transfer reactionswith enzymes and other biomolecules has made carbon nanotubes the ideal supporting material for heterogeneous catalysts. A carbon nanotube (CNT) is a tubular form of graphite sheet in nano dimensions. A single-wall carbon nanotube (SWCNT), ranging in diameter from 0.4 to >3 nm, can be visualized as formed by the rolling of a layer of graphite, called a graphene layer into a seamless cylinder. Similarly, a multiwall carbon nanotube (MWCNT), ranging in diameter from 1.4 to >100nm, can be treated as a coaxial assembly of cylinders of SWCNTs
- 6. Thesizeof a quantumdot (QD)compared to that of other materials. QD nanoparticle refers to quantum dots that have been solubilized and conjugated to affinity molecules.GFP,Green fluorescentprotein; PE,Phycoerythrin;FITC, Fluoresceineisothiocyanate.
- 7. Spectrum, absorption and emission B ri ef B a c k gr o u n d Wavelength (nm) Color Wavelength (nm) Color Examples Invisible UV Visible V I B G Y O R Invisible IR region UV/VIS Spectrophotometer works in this region
- 8. A p pl ic at io n s Aptamer/nanoparticle‐based lateral flow device. a) Adenosine‐ induced disassembly of nanoparticle aggregates into red‐colored dispersed nanoparticles. Biotin is denoted as black stars (*). b) DNA sequences and linkages in nanoparticle aggregates. Lateral flow devices loaded with the aggregates (on the conjugation pad) and streptavidin (on the membrane in cyan color) before use (c) and in a negative (d) or a positive (e) test. Exploiting properties of gold nanoparticles Purple (aggregated) Red (separated) Binding to target
- 9. Fabrication of existing biosensors by bottom‐up approach The channel region and gate dielectric of an ISFET has been fabricated by layer‐by‐layer self‐ assembly technique, (Liu and Cui, 2007). The advantages of this approach are technological simplicity and the ability to produce low‐cost sensors.
- 10. Main research activities are: 1. Design, preparation and characterization of nanomaterials for sensors and biosensors applications 2.Electrochemical sensors based on nanostructurated materials (i.e. carbon nanotubes etc.) for various monitoring and other industrial applications. 3.Nanoparticle based electrochemical detection (bio)systems and biosensors for DNA, protein and cell detection with interest for user‐friendly diagnostics, security and quality control for various applications. 4.Fast and low cost sensing devices for heavy metals and other compounds with interest for environment control and other industrial applications. 5.Lab‐on‐a‐chip systems with interest for in‐field screening of analytes and other applications.
- 11. 1. Carbon nanotubes (CNTs)combine in a unique way high electrical conductivity, highchemicalstability and extremely high mechanical strength. 2. Thesespecial properties of both single-wall (SW) and multi-wall (MW) CNTshaveattracted the interestof many researchers in the field of electrochemical sensors. 3. Here, the latest advances and future trends in producing, modifying, characterizing and integrating CNTsinto electrochemical sensing systems. 4. CNTs can be either used as single probes after formation in situ or even individually attached onto a proper transducing surface after synthesis. 5. BothSWCNTsand MWCNTscan be used to modify several electrode surfacesineither vertically oriented ‘‘nanotube forests’’ or even a non-oriented way. 6. They can be also used in sensors after mixing themwith a polymer matrix to formCNTcomposites. CARBON NANOTUBESCNTs
- 13. This nanobiosensor consists of two microelectrodes connected by a bridge of nanotubes with antibodies. Since electrical signals produced by the union of a tumour with the sensor molecule biosensor it detects the appearance of a tumour.
- 23. Integration of carbon nanotubes CNTs can be integrated into a variety of configurations to perform electrochemical detections. The current formats can be classified in groups: individual CNT configurations conventional electrodes that are modified with CNTs, in both oriented or non‐oriented configurations and, CNTs integrated into a polymer matrix, creating a CNT composite Schematicof typical CNT solubilization alternatives: (A) supramolecular wrapping with polymer; (B) CNT-Li+ conducting polyelectrolyte; (C) with amino group of 2-aminomethyl- 18-crown-6 ether; (D) by amide bonds with glucosamine; and, (E) by diamine-terminated oligomeric poly(ethylene glycol). For details, see text and Table 1.
- 24. CNTs as modifiers of electrode surfaces CNTs – both non‐oriented (random mixtures, see left) and oriented (vertically aligned) –have been used to modify several conventional electrod surfaces, glassy carbon being the most reported.
- 33. Semiconductorquantum dots The foremost application of quantum dots (QDs) as sensors is based on the Forster resonance energy transfer effect (FRET).Owing to thiseffect, thefluorescenceemanating from QDs changes from an ON state to an OFF state. FR E Ttakes place when the electronic excitation energy of a donor fluorophore is relocated to a neighbouring acceptor moleculewithout exchanging light between thedonor and the acceptor. Goldman etal. (2004), usedQDsfunctionalized with antibodies to perform multiplexed fluoroimmunoassaysfor simultaneousdetection of varioustoxins.Thistype of sensorcouldbe usedfor environmentalpurposesfor concurrentlyrecognizing pathogenslike cholera toxin or ricin in water. TheFR ETprinciple wasalsoapplied to a maltosebiosensor.Thesensingmechanismwasthe application of semiconductor QDs conjugated to a maltose binding protein covalently bound to a FR E Tacceptor dye. In absence of maltose, the dye occupied the protein binding sites. Energy transference from the QDs to the dyes quenched the QD fluorescence. When maltose was present, it replaced the dye leading to recovery of the fluorescence.
- 37. BIOSENSORS BEN213 Modification of sensor surfaces and immobilization techniques WEEK-11 SpringSemester Faculty: Asst.Prof.Dr.BetulGurunlu Faculty of Engineering & Natural Sciences Uskudar University
- 38. Chemicalmodificationsof surfacesandtheirapplications Chemical modification- two major reasons 1.to attach selective groups (binding sites or catalysts) to the sensor surface in order to recognize target species in the sample. 2.to increase the selectivity of the sensor by reduction of interfacerences arising from non-specific interactions. Surface modification- Covalent and non-covalent strategies 1.Covalent approaches- a chemical bond is made between the surface and the attached species - irreversible process. 2.Non-covalent approaches- weaker, non-bonding, interactions b/w the surface and the adsorbed species are utilized - reversible Eg., charge-charge, charge-dipole, dipole-dipole, dipole-induced dipole, and induced dipole-induced dipole interactions (van der Waals interactions) -these are less surface specfic than the covalent approaches - can be more readily achieved
- 40. Covalent modification of sensor surface The formation of a bond to some functional group on the surface. The different surface modifications are as follows: 1. Reactive organo‐silane surfaces are widely used sensor surfaces for modifcation Metal oxide surfaces were silanized followed by linking chemical groups
- 42. Cyanuric chloride activation on hydroxyl surfaces
- 43. Covalent attachment on acrylic surfaces (plastic)
- 44. Activation of carboxy surfaces by DCC
- 45. Eg., Modification of surfaces (sensor/biomolecule) through carbodiimide, avidin-biotin interactions
- 48. a.-OH surfaces are first modified with carbonyldiimidazole (CDI) to formreactive intermediate- formsa stable carbamatebond to anaminoterminated biomolecule b.-NH2 gp can be modified with glutaraldehyde which forms imine bond (Schiff's base) with an aldehyde, leaving theother aldehydefree for repeating this chemistry with an amino-terminated molecule c.Symmetric diisothiocyanates have also been used as bifunctional linkers for attachment of amine-functionalized surfaces to either thiol-terminated or amine-terminated molecules d.Heterobifunctional linker sulfosuccinimidyl-4-(N- maleimidomethyl)-cyclohexane-1-carboxylate, which first reacts with surface-bound -NH2 through displacement of the NHS, subsequently, a thol-tethered molecule can be reacted with the pendant maleimide gp. Toform surface- conjugated molecules e. EDC/NHS reaction - the most commonones
- 53. Carbodiimides
- 56. SAM
- 63. ImmobilizationTechnology Must be oriented in a specific direction ‐ immobilization technology Five major immobilization methods used in the preparation of Chemical and bio‐ sensors. 1.Covalent binding ‐ attachment of the active component to the transducer surface using a chemical reaction such as peptide bond formation or linkage to activated surface groups (thiol, epoxy, amino, carboxylic, etc) 2. Entrapment ‐ physical trapping of the active component into a film or coating. 3.Cross‐linking ‐ similar to entrapment, only a polymerization agent (such as gluteraldehyde) is used to provide additional chemical linkages between the active, entrapped component and the film or coating. 4.Adsorption ‐ association of the active component with a film or coating through hydrophobic, hydrophilic, and/or ionic interactions. 5.Biological binding ‐ association of an active biomolecule to a film or coating through specific, biochemical binding
- 64. The covalent binding method is based on the binding of enzymes and water‐insoluble carriers by covalent bonds. The functional groups that may take part in this binding are listed below: Amino group Carboxyl group Sulfhydryl group, Hydroxyl group Imidazole group Phenolic group Thiol group Threonine group Indole group Covalent attachment to a support matrix must involve only functional groups of the enzyme that are notessentialfor catalytic action. Higher activities resultfrom prevention of inactivation reactions with amino acid residues of the active sites. A number of protective methods have been devised: Covalent attachment of the enzyme in the presence of a competitive inhibitor or substrate. 1. A reversible, covalently linked enzyme-inhibitor complex. 2. A chemically modified soluble enzyme whose covalent linkage to the matrix is achieved by newly incorporated residues. 3. A zymogen precursor. Zymogen: Any of a group of compounds that are inactive precursors of enzymes and require some change (such as the hydrolysis of a fragment that masks an active enzyme) to become active
- 65. Hence, covalent binding can be brought about by the following: Diazotization : Amide bond formation : Alkylation and Arylation: SUPPORT--N=N--ENZYME. SUPPORT--CO-NH--ENZYME SUPPORT--CH2-NH-ENZYME SUPPORT--CH2-S--ENZYME SUPPORT--CH=N--ENZYME SUPPORT--CNH-NH--ENZYME SUPPORT--S-S--ENZYME Schiff's base formation : Amidation reaction : Thiol-Disulfide interchange : UGI reaction Gamma-Irradiationinduced coupling Carrier binding with bifunctional reagents : SUPPORT-O(CH2)2N=CH(CH2)3 CH=N-ENZYME
- 68. Glutaraldehyde- The surfaces (proteins) of microorganisms are linked with the surfaces of other microorganisms by aldehyde groups of glutaraldehyde
- 71. Entrapmentand encapsulation The immobilized enzyme can be classified into four types: particles, membranes, tubes, and filters. Most immobilized enzymes are in particle form for ease of handling and ease of application. Particles - The particle form of immobilized enzyme on solid particles. Membranes - Enzyme membranes can be prepared by attaching enzymes to membrane- type carriers, or by molding into membrane form. The molding is done after the enzymes have been enclosed within semi-permeate membranes of polymer by entrapment. Tubes- Enzyme tubes are produced using Nylon and polyacrylamide tubes as carriers. The polymer tube isfirst treated ina seriesof chemicalreactionsand theenzymeisboundby diazo coupling to give a tube in a final step. Fibers - Enzymes that have been immobilized by entrapment in fibers to form enzyme fibers. The solid supports used for enzyme immobilization can be inorganic or organic . Some organic supportsinclude: P olysaccharides, Proteins, Carbon, P olystyrenes, P olyacrylates, MaleicAnhydride basedCopolymers,Polypeptides,Vinyl andAllyl Polymers,and Polyamides.
- 72. ImmobilizationthroughEntrapment The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. It is done in such a way as to retain protein while allowing penetration of substrate. It can be classified into lattice and micro capsule types
- 73. Cross‐linking is based on the formation of chemical bonds, as in the covalent binding method. The immobilization is performed by the formation of intermolecular cross‐ linkages between the enzyme molecules by means of bi or multifunctional reagents. The most common reagent used for cross‐linking is glutaraldehyde. Cross‐linking reactions are carried out under relatively severe conditions. These harsh conditions can change the conformation of active center of the enzyme; and so may lead to significant loss of activity. The carrier‐binding method is the oldest immobilization technique for enzymes. In this method, the amount of enzyme bound to the carrier and the activity after immobilization depend on the nature of the carrier. The following picture shows how the enzyme is bound to the carrier: The selection of the carrier depends on the nature of the enzyme itself, as well as the: Particle size Surface area Molar ratio of hydrophilic to hydrophobic groups Chemical composition ‐polysaccharide derivatives such as cellulose, dextran, agarose, and polyacrylamide gel.
- 74. Physical Adsorption Mode Based on the physical adsorption of enzyme protein on the surface of water-insoluble carriers. Theprocessesavailable for physical adsorption of enzymesare: StaticProcedure Electro-deposition Reactor Loading Process Mixing or Shaking Bath Loading Adsorption tendsto be lessdisruptive to theenzymaticprotein thanchemicalmeansof attachment because the binding is mainly by hydrogen bonds, multiple salt linkages, and Van der Waal's forces. Becauseof theweak bondsinvolved,desorption of theprotein resulting from changesin temperature, pH,ionicstrengthor eventhemerepresence of substrate, is often observed. Another disadvantage is non-specific, further adsorption of other proteins or other substances as theimmobilized enzymeisused.Thismayalter the properties of the immobilized enzyme or, if thesubstanceadsorbed isa substratefor theenzyme, the rate will probably decrease depending on the surface mobility of enzyme and substrate. Adsorption of the enzyme may be necessary to facilitate the covalent reactions described later inthispresentation. Stabilization of enzymestemporarily adsorbed ontoa matrix hasbeen achieved by cross-linkingtheprotein in a chemical reaction subsequent to its physical adsorption.
- 75. Site-specific chemical modification of biomolecules