1 3.051J/20.340J Lecture 17 Biosensors 1. What are biosensors? The term is used in the literature in many ways. Some definitions: a) A device used to measure biologically-derived signals b) A device that “senses” using “biomimetic” (imitative of life) strategies ex.,“artificial nose” c) A device that detects the presence of biomolecules We will adopt a recent IUPAC definition: “A self-contained integrated device which [sic] is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element which is in direct spatial contact with a transducer element.” 2. Uses of biosensors • Quality assurance in agriculture, food and pharmaceutical industries ex. E. Coli, Salmonella • Monitoring environmental pollutants & biological warfare agents ex., Bacillus anthracis (anthrax) spores • Medical diagnostics ex., glucose • Biological assays ex., DNA microarrays2 3.051J/20.340J 3. Classes of biosensors A) Catalytic biosensors: kinetic devices that measure steady-state concentration of a transducer-detectable species formed/lost due to a biocatalytic reaction Monitored quantities: i) rate of product formation ii) disappearance of a reactant iii) inhibition of a reaction Biocatalysts used: i) enzymes ii) microorganisms ii) organelles iv) tissue samples B) Affinity biosensors: devices in which receptor molecules bind analyte molecules “irreversibly”, causing a physicochemical change that is detected by a transducer Receptor molecules: i) antibodies ii) nucleic acids iii) hormone receptors Biosensors are most often used to detect molecules of biological origin, based on specific interactions.3 3.051J/20.340J Transducer biological elementElectrolyte Analyte (ex., blood) Signal (1)(2) 4. Biosensor Components Immobilized (electrochemical) (chemical target) External medium Semipermeable membranes Analyte: chemical/biological target Semipermeable Membrane (1): allows preferential passage of analyte (limits fouling) Detection Element (Biological): provides specific recognition/detection of analyte Semipermeable Membrane (2): (some designs) preferential passage of by- product of recognition event Electrolyte: (electrochemical-based) ion conduction medium between electrodes Transducer: converts detection event into a measurable signal3.051J/20.340J 4 A) Detection Elements 1) Catalysis Strategies: enzymes most common ex., glucose oxidase, urease (catalyzes urea hydrolysis), alcohol oxidase, etc. Commercial Example: glucose sensor using glucose oxidase (GOD) Glucose + O2 + H2O → Gluconic acid + H2O2 GOD 3 potential measurement routes: 1. pH change (acid production) 2. O2 consumption (fluorophore monitor) 3. H2O2 production (electrochemical) Commercially Available Biosensors: glucose, lactate, alcohol, sucrose, galactose, uric acid, alpha amylase, choline, L-lysine—all amperometric based (O2 /H2O2) 2) Affinity Binding strategies: antibodies & nucleic acid fragments most common Commercial Example: DNA chip53.051J/20.340J B) Transducers 1) Electrochemical: translate a chemical event to an electrical event by measuring current passed (amperometric = most common), potential change between electrodes, etc. Oxidation reaction of the reduced chemical species Cred: Cred → C + ne− ox Amperometric Devices Measured current is mass transport limited Cred * Cred δ x (distance from electrode) ℑ = 96,487 coulombs (Faraday const.) = niilim =− ℑ AJ C where J is the flux: red − 0J =−D dCred ≈−D * δ A = electrode area δ= boundary layer width nADCred ⇒ i ≈ℑ * dx δ6 3.051J/20.340J Example: Glucose sensor based on oxidation of peroxide (most commercial devices) Gel incorporating glucose oxidase + Electrolyte Au working Au counter electrode electrode Glucose + O2 + H2O → Gluconic acid + H2O2 GOD Anodic: H2O2 → O2 + 2H+ + 2e-current passed thru working electrode (Recall: oxidation occurs at anode; here, O-1→O0)7 3.051J/20.340J 2) Photochemical: translate chemical event to a photochemical event, measure light intensity and wavelength (λ) a) Colorimetric: measure absorption intensity Examples Indirect: H2O2 + Dye Precursor Colored Dye peroxidase enzyme Direct: flavin adenine dinucleotide (FAD) bound cofactors (redox sites on GOD) absorption at 377nm & 455nm disappears in presence of glucose b) Fluorescence Example 1: DNA microarrays– fluorophores selectively bound to detected molecule via avidin-biotin complex; commercialized by Affymetrix (S. Fodor)8 3.051J/20.340J Example 2: fiber optic sensors: fluorophores incorporated into tip change fluorescence level depending on level of target present crosslinked network tip with entrapped fluorophores & E=hν2 light of initial E = hν1 biomolecule (light used to photopolymerize matrix!) measure fluorescent light returning: excites fluorophore Typically: - Oxygen present at tip quenches fluorescence from trapped fluorophore (ex., tris(4,7-diphenyl-1,10 phenantroline) Ru(II) dichloride = Ru(dpp)32+ Cl2) - Action of trapped oxidase (biological element, ex., GOD) depletes O2, causing ↑ fluorophore emission Glucose + O2 + H2O → Gluconic acid + H2O2 GOD How can we account for natural O2 fluctuations? Multichannel fiber optic: 1. enhancing selectivity and/or 2. multianalyte detection How can we measure multiple analytes? MD. Marazuela et al., “Fiber-optic biosensors- an overview”, Anal. Bioanal. Chem. 372, 664 (2002).9 3.051J/20.340J Example 3: Semiconductor nanoparticles (quantum dots) currently in development, ex., Quantum Dot Corp. (P. Alivasatos) ADVANTAGES:CdSe Typically affinity binding-based tethered antibody i) QD band gap (and hence emission) varies with size ⇒ multiple analyte capability 2 nm CdSe⇒ green 5 nm CdSe⇒ red ii) sharp, intense emission spectra (higher signal/noise) Intensity λ iii) can be used for surface or solution-based approaches A.P. Alivisatos, Science 271, 2013 (1998).103.051J/20.340J c) Reflectance Example 1: “Nanobarcodes” – reflection from surface of multilayer metallic rods provides optical signature; being developed by Surromed, Inc. (M. Natan) Affinity-binding based 0.04 – 15 µm Al2O3 membrane (dissolve w/NaOH) Ag electrode 20-500 nm (dissolve w/HNO3) Pd Au Ag Au Pt Made by electrochemical reduction of a series of metal salts into template pores Reflectance microscopy gives unique