Biosensors and Biochips: Principles, Fabrication and Applications Biosensors A biosensor is defined by IUPAC (1997) as: ‘‘A device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals’’. Almost all biosensors are based on a three-component system: a support, a biological recognition element (ligand) that facilitates specific binding or biochemical reaction of a target, and a signal conversion unit (transducer). Biosensors Miners used birds to reveal the presence of toxic gases in mines. Birds like canaries die if exposed to low gas concentrations. This is one early example of a disposable ON/OFF biosensor. The gas is the analyte, the canary acts as the biological active species, and the detection limit is the minimal gas concentration that can kill the bird. Gas concentration is below the detection limit The bird lives Gas concentration is above the detection limit The bird dies Biosensors An even earlier example of disposable ON/OFF biosensor is the food taster, usually a slave that tested the king food for poisons (like Remy in Ratatouille). Of course, the food taster was disposed if deadly poison was present in the food. Biosensors A more recent example of disposable ON/OFF biosensor is the mine detector, a rat trained to find mines. Its light weight allows it for walking on mines without any risk of explosion. Of course, the mine tester is disposed if the mine is too sensitive to pressure. Biosensors – History 1956 Clark published a paper on the oxygen electrode, and used the term “enzyme electrode”. The concept of biosensor was born. 1969 Guilbault and Montalvo described the first potentiometric enzyme electrode, based on the use of urease to detect urea. 1975 The Yellow Springs Instrument Co. launched a glucose biosensor based on the amperometric detection of hydrogen peroxide. 1976 The BIOSTATOR (electrochemical glucose biosensor in artificial pancreas) was invented. 1982 The first needle-type enzyme electrode for subcutaneous implantation of glucose biosensors was reported. 1987 MediSense launched a pen-sized meter for home blood- glucose monitoring. Biosensor requirements To satisfy a large number of applications, a biosensor should be: easy to manufacture in large numbers easy to use cheap if disposable reusable if expensive fast in response able to perform multiple tests at the same time (microarray) Biosensor Structure Almost all biosensors are based on a three-component system: a support, a biological recognition element (ligand) that facilitates specific binding or biochemical reaction of a target, and a signal conversion unit ( transducer). Biosensors – Transducer The transducer converts the biochemical interactions into a measurable electronic signal. Electrochemical, electrooptical, acoustical, and mechanical transducers are among the many types found in biosensors. The transducer works either directly or indirectly. Direct Transducers Direct detection sensors, in which the biological interaction is directly measured in real time, typically use non-catalytic ligands such as cell receptors or antibodies. The most common direct detection biosensor systems employ surface plasmon resonance . Quartz resonator transducers that measure changes in acoustic resonance and opto– mechanical biosensors ( microcantilevers) are other direct recognition sensors. Diode array Laser Prism θ Metal Polymer Flow cell [...]... Binding to Substrates O HN biotinylated antibody NH H H OH S biotin O Biosensor Fabrication Many high-level technologies have been developed and consolidated for microelectronic industry, and can be used for the production of micro- and nano-scaled biosensors Screen printing Liquid-handling techniques (syringe-type processes, ink-jet printing, etc.) Photolithography Nanopatterning Screen Printing or... because of high specificity, versatility, and strong binding to the antigen Aptamers, protein-binding DNA or RNA (Kd 108–109 M) amenable for high-throughput screening, are easy to manufacture However, they can only be used for targets that bind nucleic acids Peptides, combined binding agents derived from low-affinity ligands and combinatorial chemistry ligands for biosensors Substrates Silicon wafers... electroactive compound on the secondary ligand Fluorescence, measuring fluorescence of the secondary ligand Fluorescence Resonance Energy Transfer (FRET), a process where energy from an excited fluorophore is transferred to a neighboring acceptor molecule Biosensors – Ligands The most important characteristics for ligands are affinity and specificity Ligands can be: Molecules, macromolecules, whole... Syringe–type Processes: Pin printing Ink-jet Printing: Piezo Inkjet Ink-jet Printing: Piezo Inkjet Accuracy of dispensing No contamination Overprint possible Dispensing on non-flat surfaces Viscosity Multi reagents logistics Risk to denaturation Long process time Ink-jet Printing: Thermal (Bubble) Inkjet Ink-jet Printing: Thermal (Bubble) Inkjet Speed, volume and ejection frequency of the drops are... determined by the ejector system geometry and by ink fluidodynamics Liquid drops 200 – 2 pl, diameter on paper 300 m – 2 0-3 0 m The overheated region is just over the resistor (several m thick) and do not affect the bulk liquid ejected Accurately metered quantity, high reproducibility No appreciable variation in biomolecules properties after printing Precise and clean process (no intermediate washing... Mechanism – CH2 -CH H+ hν SbF6 H+ –SbF6 Δ n CH2 -CH CH3 O C O C CH3 CH3 O n Mass S+ Resist Components • Polymer • Solvent • Photoacid Generator (PAG) • Additives (e.g DI, plasticizer) OH + H3C CO2 + with acid without acid C CH2 H3C + H+ 50 100 150 200 250 Temperature (°C) 300 350 Microcontact Printing Si pour PDMS prepolymer PDMS Si cure, peel off PDMS Simple Mass produceable Flat and curved surfaces... for very expensive fluids) Clogging – Kogation ( dedicated printing buffers) Photolithography Chemically Amplified Photoresist Resist Coat & Bake Wafer Mask Expose (157 or 193 or 248 nm) Positive Post-Exposure Bake (PEB) Develop Etch (Plasma) Strip Negative Photoresist Photosensitive polymer used for transferring pattern to substrate Has to: ! Adhere to substrate ! Undergo radiation induced solubility... on hydrophilic surfaces COVALENT IMMOBILIZATION The surface must be functionalized; the functional groups can be separated from the surface using a spacer; the functional groups can be selective or non-selective Both methods can be performed either directly on the support surface or on an additional layer deposited on the support Proteins on Surfaces Polar residue Non–Polar residue Positively charged . Biosensors and Biochips: Principles, Fabrication and Applications Biosensors A biosensor is defined by IUPAC (1997) as: ‘‘A device. transferred to a neighboring acceptor molecule. Biosensors – Ligands The most important characteristics for ligands are affinity and specificity. Ligands can be: Molecules, macromolecules,. pressure. Biosensors – History 1956 Clark published a paper on the oxygen electrode, and used the term “enzyme electrode”. The concept of biosensor was born. 1969 Guilbault and Montalvo