Biossensores Lecture Notes 2024 PDF

Biossensores Aula 2 Optical, Chemical and Mechanical Biosensors Examples of Microfabrication Hugo Águas - FCT-UNL 2024 1 BIOSENSORS Definitions: Device used to measure relevant biological i...

Biossensores Aula 2 Optical, Chemical and Mechanical Biosensors Examples of Microfabrication Hugo Águas - FCT-UNL 2024 1 BIOSENSORS Definitions: Device used to measure relevant biological information Device that uses a biological component as part of a transduction mechanism: Antibodies Enzymes DNA, RNA cells Organs / Systems What is a Biosensor? Settings: It can be developed from any sensor by adding a biological component. Transduction: Electric optics mechanics Thermal Chemistry Magnetic 2 BIOSENSORS Biosensors..... Solid State Sensors Optical Sensors: Use the modification of light intensity or its polarization for detection Chemical Sensors: They use changing the chemical properties (pH, concentration,...) of the medium for detection Mechanical Sensors: They use changing the physical properties (pressure,...) of the medium for detection Actuators Solid State Micro-actuators Mechanical Actuators: They use small actuators (eg cantilevers) to act in the middle MEMS: Integration of Solid State Sensors and Micro-actuators in a single device 3 Optical Sensors – Evanescent Field Sensor ATR – Working principle 4 Optical Sensors – Evanescent Field Sensor ATR – Working principle 5 Optical Sensors: Evanescent Field Sensors - SPR SPR sensor (surface plasmonic resonance): Optical phenomenon that allows the detection of refractive index changes at the separation interface between a metal and a dielectric. Light incident on the metal-dielectric interface causes a surface plasmon to be excited at a certain angle of incidence of the light beam (called the resonance angle). The excitation of the surface plasmonic causes a very large decrease in the metal's reflectivity, as the incident photons are absorbed by the surface plasmon. The resonance angle strongly depends on the refractive index of the medium next to the metallic film, which is proportional to the concentration of the element to be measured. Optical Sensors: Evanescent Field Sensors - SPR SPR Sensor: It generally consists of an Au film (≈400Å) deposited on a transparent substrate with a high refractive index (eg TiO2 ). A film is placed over Au with the capacity to absorb the “anchor” receptor molecules, which are normally the complementary pair of the molecule to be detected and to which they will bind. The bond causes a change in the refractive index with the consequence of a change in the resonance angle, Probe Target Optical Sensors: Evanescent Field Sensors - SPR Example of a measurement sequence - Sensogram Optical Sensors– Evanescent Field Sensors in Optical Fibers Working Principle: They are based on the principle of evanescent field modulation. In a waveguide, light is transmitted under conditions of total internal reflection. The electromagnetic field has an exponential decay in the areas surrounding the core, generating the so-called evanescent field. Any molecular interaction that occurs within the evanescent field generates changes in the characteristics of the light propagated through the waveguide. Sensores Ópticos – Sensores de Campo Evanescente Promenor da micromaquinação da fibra óptica Optical Sensors– Evanescent Field Sensors in Waveguides Principle of measurement: A film of selective receptor molecules is immobilized on the sensor surface; Depending on the selectivity of the film, it is possible to detect proteins, DNA and environmental contaminants, among others 12 Chemical Sensors - ISFET type sensor ISFET sensors were discovered in 1970 and were the first in this class to have the chemically sensitive layer integrated into solid-state electronics. The operating principle is to replace the traditional gate of MOSFETs with a gate that is sensitive to ions (for example palladium or iridium). Polar species charges are absorbed between the gate and the insulator, causing a corresponding charge to appear on the semiconductor surface. As a result, the capacity- voltage curve and the IDS versus VG curve will shift along the voltage axis. Voltage displacement is thus a measure of the concentration of absorbed species. Alternatively, the gate metal can be replaced with an ion selective membrane. Chemical Sensors - ISFET type sensor 15  Chemical Sensors - ISFET type sensor Device Fabrication PECVD ▪PASSIVATION 50 nm n+a-Si:H @ 250ºC (PECVD) 100 m ▪150 150250 7059nm nm a-Si:H AlSiN @ 250ºC source-drain Corning nm xglass @ 350ºC contacts ▪ 100 nm SiNx @ 350ºC 20 m Top View Metal “top-gate” TFT Electrolyte-gate a-Si:H TFTs 150 nm Al / 15 nm TiW 50 nm SiO2 SiNx exposed Chemical Sensors - ISFET type sensor Size of some ISFET type sensors: ISFET pH meter Nanowire-based sensors 18 Nanowire-based sensors 19 Mechanical Sensors - Sensors based on silicon structures Mechanical sensors based on silicon micro tips: The operating principle of this type of sensor is similar to that of the AFM, using a silicon microtip with dimensions of 100 μm in length, 10 μm in width and a thickness of less than 1 μm. These tips are sensitive enough to measure the force carried by just the bond between two complementary biological molecules. To do this, a biological molecule is immobilized on the tip and its complement on the surface of a substrate Mechanical Sensors - Sensors based on silicon structures Mechanical sensors based on silicon micro tips (MEMS): Alternatively, biological molecules such as antibodies can be immobilized on the surface of the tip, which will specifically bind to that substance to be detected. When the connection is made, a response of the nanomechanical type is produced at the tip of the sensor, which consists of a variation of the deflection and/or the resonant frequency. The response is measured by an optical system that consists of a laser beam that focuses on the final surface of the microtip, being reflected towards a position sensor. Examples of tips: Bending Vibration Mechanical Sensors - Sensors based on silicon structures Detection by cantilevers Microcantilever technology: a) with immobilised protein for a specific bacterium b) bending after adsorption of bacteria to the protein Nanomechanical oscillator Use of "cantilevers" to detect viruses and bacteria. The tip can be coated with antibodies specific for a specific virus and in contact with the substance they are attracted. Tip wobble can be measured and compared before being exposed to that same substance. http://pubs.rsc.org/en/content/articlehtml/2007/LC/B707401H 23 Problem A cantilever was placed in a solution containing “E. coli bacterium". After being removed, the vibration frequency was measured and compared with the initial one. How 5 E. coli many individual E. coli bacterium cells cells were left in the cantilever? data:  w1 = 4.6 kHz => mE. coli cell = 665 x 10-15 grams  w2 = 23 kHz => mE. coli cell = 3325 x 10-15 grams 24 Transducers-Mass based methods Detection by cantilevers (a) A mechanical cantilever resonator containing an embedded microfluidic channel. (b) Decrease in resonant frequency as the density inside the embedded channel increases. (c) Frequency modulation by single particle movment 25  Mechanical Sensors - Sensors based on silicon structures Manufacturing Process of a Micro-bridge Thin film technology, T ≤ 110 ºC Surface micromachining MEMS Technology 7 - Sacrificial layer removal 6 - SiO2 layer patterning 200nm 5 - Al deposition and bridge definition 4 - Structural layer deposition (n+-a-Si:H) 300nm 3 - Sacrificial layer (PR) patterning 1m 2 - Al gate electrode 100nm 1 - Glass substrate Teresa Adraga et.al. ICANS 21 Mechanical Sensors - Sensors based on silicon structures O Oxidation OH Silanization O Si NH2 SiO2 OCH2 CH3 O O O (Cholic acid, OH Na+O- - S O O N N 12hr, RT) H2 N Si OCH2 CH3 O O O O OCH2 CH3 surface (Sulfo-EMCS, 2 hr) (2 hr, APTES) O O S-5´-DNA probe-FITC-3´ O O O Si NH N N SiO2 O Si NH DNA O O O O Immobilization Functionalized DNA immobilization surface Teresa Adraga et.al. ICANS 21 DNA= 30 pmol/ cm2 Mechanical Sensors - Sensors based on silicon structures Resonance Frequency Measurements Electrostatic actuation Optical detection in vacuum initial state Si photodetector red diode laser spot size  50 m measure functionalization measure V= VDC + VAC sin (2f t) DNA immobilization measure DNA hybridization Microfabrication Techniques– R. I. Etching de a-SiC:H Gases : CHF3 + O3 (50%) Pressure : 100 mTorr r.f. Power : 100 W Etching Rate : 268 A/min a-SiC:H a-SiC:H Micro-Tunnels Mechanical Micro-Actuators - Based on Silicon Structures ⚫ a-SiC:H micro bridges X thermal actuation processing : front side Si bulk micromachining (1.5 hr,KOH, 80 oC) heating element : evaporated Aluminum PECVD based process : SiOxNy as masking and protective material limited movement Mechanical Micro-Actuators - Based on Silicon Structures MEMS structures consisting of cantilevers of a-SiC:H and a-SiC:H / SiOxNy. – Can function as mechanical actuators ▪ 100 m x 300 m ▪ 100 m x 500 m Two type Mechanical Micro-Actuators - Based on Silicon Structures Film manufacturing conditions ⚫ Standard r.f. PECVD (13,56 MHz) SiH4 CH4 N2 O r.f. Sample Temp. Flow Flow Flow Power Type (ºC) (sccm) (sccm) (sccm) (W) a-SiC:H 3.6 14.0 - 20 320 x ~ 0.5 320 x ~ 0.5 SiOxNy 15.0 - 37.5 200 y ~ 0.15  Mechanical Micro-Actuators - Based on Silicon Structures Fabrication Steps SiOxNy (1.5 m) Cr (0.3 m) a-SiC:H (1 m) Si (100) Etching of SiOxNy - (HF) Etching of a-SiC:H - (CHF3+O2) Micromachining (etching) Si (KOH, 1.5 hr) Etching of SiOxNy (HF) Mechanical Micro-Actuators - Based on Silicon Structures SiOxNy (1.5 m) Fabrication Steps Cr (0.3 m) SiOxNy (0.5 m) Cr (0.3 m) a-SiC:H (1.5 m) Si (100) Etching of SiOxNy - (HF) Etching of a-SiC:H - (CHF3+O2) Micromachining (etching) Si (KOH, 1.5 hr) Etching of SiOxNy (HF) Mechanical Micro-Actuators - Based on Silicon Structures Thermally Actuated Cantilevers Mechanical Micro-Actuators - Based on Silicon Structures Thermally Actuated Cantilevers Mechanical Micro-Actuators - Based on Silicon Structures a-SiC:H / SiOxNy a-SiC:H Cantilevers Cantilevers Thermally Actuated Cantilevers Mechanical Micro-Actuators - Based on Silicon Structures a-SiC:H / SiOxNy a-SiC:H Cantilevers Cantilevers Thermally Actuated Cantilevers