Biosensors Flashcards
Bionsensors
The aim of biosensors is to produce an electrical or optical signal proportional to the concentration of a specific chemical (analyte) or a set of chemicals.
Regarding selectivity, different types of biomolecules have the highly remarkable capability of molecular recognition and show a strong affinity to the constituents in the analyte or sample. Some couples are particularly important, for example, enzyme/substrate, antibody/antigen and nucleic acids/complementary sequences, in the construction of a sensing layer or in establishing intelligence in the sensors. In addition to these, micro-organisms, animal or plant whole cells and even tissue slices can also be incorporated in the sensing layer.
Enzymes: highly specialized proteins specifically able to catalyse metabolic reactions in living organisms.
Antibodies: are naturally produced both by animals and human beings and are able to react against foreign substances.
Bioreceptor and transduction
In a biosensor, the phenomenon is recognized by a biological system called a bioreceptor which is in direct contact with the sample and forms the sensitive component of the biosensor.
In another words, a bioreceptor has a particularly selective site that identifies the analyte.
So, the function of a biosensor is to transform a biological event into an electrical (optical) signal. This sensing ability is dependent entirely on the intelligent material of which it is made up, because the lesser the interference allowed by the intelligent material, the better the selectivity of the sensor.
Thus, the bioreceptor has the primary importance in selectivity.
There are two classes of bio-recognition processes, bio-affinity recognition and bio-metabolic recognition, which are differentiated by the general method of detection.
Both of them involve the binding of a chemical species with another which has a
complementary structure => shape-specific binding.
Bio-affinity recognition:
• Very strong binding.
• The transducer must detect the presence of the bound receptor-analyte pair (for example, antibody-antigen).
Bio-metabolic recognition:
• After binding, the analyte and any other co-reactants are chemically altered to form product molecules.
• The transducer must detect the change in the concentration of either the products or the co-reactants, the pH, or the heat liberated (or light emitted) during the reaction (for example, enzyme-substrate reactions or metabolism of specific molecules by tissues and cells).
Immobilization of biological elements
The immobilization of the biological element on the physical transducer is one of the keys to a high sensitivity, long-lived biosensor. The immobilization must:
• confine the biologically active material on the transducer and keep it from leaking out over the lifetime of the biosensor;
• allow contact to the analyte solution;
• allow any products to diffuse out of the immobilization layer;
• not denature the biologically active material: critical requirement, since enzymes, antigens, cells and tissues are all fragile biological materials that can be easily rendered inactive by mechanical damage, heat or freezing, chemical toxins, lack of certain
chemicals, …
There are two basic types of immobilization techniques that depend on the basic mechanics of
the immobilization:
• BINDING: involves attaching the biologically active material directly to the surface of the transducer, or the surface of a base membrane on the transducer (adsorption and covalent binding);
• PHYSICAL RETENTION: involves separating the biologically active material from analyte solution with a layer on the surface of the transducer, which is permeable to the analyte and any products of the recognition reaction, but not to the biologically active material (membrane confinement and matrix entrapment).
Membrane confinement
It is the most straightforward physical retention immobilization technique: you entrap a solution containing the biologically active material on the surface of the transducer using a semipermeable membrane. Pores must be large enough to let the analyte, the products and the solution through, but small enough to retain the biologically active material. Usually polymers of polyamide or polyether sulfon, or dialysis membrane. The membrane must be matched to the biosensor so that it does not appreciably affect response of the transducer.
Matrix entrapment
It involves the formation of the porous encapsulation matrix around the biologically active material (enzyme, antigens or whole cells). It is accomplished by either cross-linking a mixture of the matrix forming material and the biologically active material or through the formation of a gel containing the biologically active material. The matrix must have pores large enough to let the analyte, the products and the solution through, but small enough to retain the biologically active material. Natural materials are the easiest to use as the matrix, since they are not toxic to the biological materials. The number of synthetic polymers that can be used is limited by the toxicity of the monomers, cross-linking agents, and by-products of the polymerization reaction, as well as the polymerization conditions.
Adsorption
It is the simplest method of immobilization: the transducer surface is exposed to a solution of the biologically active material for a period of time, then the surface is washed to remove the loosely bound material and the biosensor is ready to be used. Biologically active material hold on the surface by a combination of van der Waals, hydrophobic and ionic forces or hydrogen bonds (this variety is due to complexity of biological materials so different parts of the molecule or structure are attracted to the
surface by different forces).
• The advantage, besides its ease of use, is that forces are relatively “gentle”, not denaturating the material.
• The disadvantage is that forces are “gentle”, so molecules are weakly bound to the transducer. A change in temperature, analyte concentration, pH, or ion concentration can desorb the biologically active material. Another operational problem is that it is
difficult to quantify the amount of biologically active material that has been adsorbed on the transducer.
Covalent bonding
It is a more permanent binding of the biological material. The surface of the transducer or of the
membrane on the transducer must be treated to have reactive groups to which the bioactive material can bind. The surface treatment must have two terminal functional groups: one which binds covalently to the transducer and one that binds to
the biologically active material =>highly asymmetric molecules.
The biologicaly active material is directly on the surface of the transducer, reducing the response time of the biosensor since the diffusion time of the products of the reaction to the transducer is reduced
The bond to the transducer is much stronger than physical adsorption, so the sensor lifetime is longer but covalent binding may chemically modify the important binding sites on the biological material and denature the molecules.
Transduction principles
The transducers may take different forms depending on the specific application, but so far the emphasis has been on the following electronic configurations:
(1) Optoelectronic detectors (optical);
(2) Field-effect transistors (potentiometric);
(3) Amperometric electrodes (electrochemical);
(4) Thermistors/Thermocouples (calorimetric).
(5) Microgravimetric devices (mechanical)
To obtain a better electrical signal, bioreceptor and transducer have to be compatible with each other; for example, electrochemical transducers couple relatively easily with enzymes because they have reasonable biocompatibility with each other.
The ISFET
In an ISFET biosensor, an ion-sensitive membrane and an immobilized enzyme membrane are formed on the gate region (ENFET).
Enzyme-immobilized membrane deposition on the ISFET can be accomplished by the integrated circuit fabrication methods, either by spin coating or lift-off methods, or by ink-jet printing.
Moreover, there are attempts to produce multifunction-biosensors by utilizing the ISFET’s characteristics. A single-chip multibiosensor, which is capable of simultaneously measuring urea, glucose and potassium, was manufactured by using three kinds of integrated ISFET on a sensor chip.
Since ISFET biosensors are produced by the integrated circuit production method, they could be reduced in size and mass-produced. It is expected that they will be used in various fields, some of the more important applications being clinical diagnosis, the monitoring of artificial dialysis and blood glucose control in diabetes.
Most of the sensors currently in medical use are enzyme sensors.
Amperometric biosensors
Amperometric biosensors measure the concentration-dependent current through an electrochemical electrode coated with the biologically active material.
Amperometric transduction is based on the oxidation or reduction of an electroactive species on an electrode surface. The biologically active material, usually an enzyme, oxidizes or reduces the analyte or the enzyme’s substrate.
With amperometric sensors, the electrode potential is maintained at a constant level sufficient for the oxidation or reduction of the species of interest (or a substance electrochemically coupled to it); the current flowing is then proportional to analyte concentration.
These systems are capable of giving electrical signals in microA range within 30 seconds and are capable of detecting nanomolar-to-micromolar quantities of reactants.
Typical substrates used in amperometric biosensors are glucose, urea, cholesterol, fructose, sucrose and ethanol, as well as tissue and whole organisms.
Optical biosensors
The typical structure of a biosensing device which exploits light intensity is that of a pH or ion-selective dye immobilized behind a membrane on the end of an optical fibre. The sensing is established by monitoring the changes in the intensity of light emitted by the dye on binding the analyte with a light-emitting diode.
Another optical biosensor developed exploits the evanescent wave component of a light beam that is internally reflected within the optical fibre. The sensing principle depends on immobilized anti-bodies on the surface of the optical fibre and on binding to corresponding antigens, the light internally reflected is reduced in intensity. Hence, the level of binding can be assumed.
Thermal biosensors
Thermal detection measures the enthalpy of the detected reaction. The transducers are usually thermistors or thermocouples.
The enzymes or other biological recognition elements are immobilized on supports and the analyte solution flows through a heat exchanger to set its initial temperature. Then the peak signal is proportional to the concentration of the analyte.
The total heat evolved in a chemical reaction is proportional to the molar enthalpy of the reaction: Q=-n DeltaH where Q is the total heat evolved in the reaction, n is the number of moles of the product formed, and DeltaH is the molar enthalpy change. The temperature change of the system, including the solution, is given by DeltaT=Q/Cs=-n DeltaH/Cs
where CS is the heat capacity of the system.
Enzyme reactions usually evolve a relatively large amount of heat and, thus are well suited for calorimetry.
-PROBLEMS: the susceptibility of the transducer to environmental temperature variations, interfering
chemical reactions and processes, such as mixing and adsorption, which give off or absorb heat.
-ADVANTANGE: wide range of linear response, about 5 orders of magnitude
Mass detection biosensors
Microcantilever: by shrinking the cantilever structure to a microscopic (or nanoscopic) size one obtains both a low spring constant (i.e. high sensitivity to applied forces or stresses) and a high resonant frequency, for fast response times and high immunity to external mechanical noise. Measurements in the 10-14 N range at the level of single bio-molecular pairs are possible.
Arrays of cantilevers
STATIC MODE MICRO AND NANO BALANCES
-Cantilever functionalised on one side with a probe molecule (DNA oligomer, antibody, …)
-Target molecule (complementary DNA oligomer, antigene, …) selectively binds to the probes
-Deflection of the cantilever due to increased stress
-Readout technique: optical lever (laser + alignment set-up + photodiode) :
° nanometer sensitivity
° no fluorescent tags needed
° implementation of redundancy (several parallel cantilevers or reference cantilever to null common
mode effects like T variations)
° ne side functionalization not so easy (ink-jet derived techniques)
DYNAMIC MODE
- Cantilever functionalised on both sides with a probe molecule (DNA oligomer, antibody, …)
- Target molecule (complementary DNA oligomer, antigene, …) selectively binds to the probes
- Change of resonant properties (f0 and Q) due to increased mass
- Readout technique: OPTICAL LEVER
- Femtogram (at least) sensitivity: since frequency change can be measured to very high precision (Deltaf/f < 10^-5 to 10^-6 ), very small mass changes can be measured –> high sensitive biosensors –>better sensitivity with respect to the static mode –>theoretically zeptogram (10-21 g) is achievable
- No fluorescent tags needed
- Implementation of redundancy (several parallel cantilevers or reference cantilever to null common mode effects like T variations)
- Two sides functionalization is easier (simple dipping) … apparently …
Packaging of biosensors
FUNCTIONS OF BIOSENSORS PACKAGING:
- To protect the sensor from the environment: the sensor can perform its function properly and within the designated lifetime.
- To protect the environment from the sensor material and operation: biocompatibility and no toxic products delivered in the environment.
The protection of the sensors from degradation or breakdown in their environment include:
- Electrical isolation or passivation of leads and electronics from ions and moisture that causes conductive paths for leakage currents.
- Mechanical protection to ensure the structural integrity and dimensional stability.
- Optical and thermal protection, to prevent undesired effects of ambient light and heat that may alter the signal and sensor operation.
- Chemical isolation of the sensor from the harsh chemical environment
The protection of the environment from the sensor may include:
- Sensor materials selection to eliminate or reduce body reaction.
- Sensor mode of operation and packaging selection to avoid toxic products or effects. ù
- The sterilization of the sensors.
