Electrochemical sensors, biosensors, and their biomedical applications
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AB - This chapter focuses on the various developments in electrochemical immunoassays and immunosensors after Recent developments in electrochemical immunoassays and immunosensors Jeremy M. Department of Molecular Sciences. Abstract This chapter focuses on the various developments in electrochemical immunoassays and immunosensors after Fingerprint Immunosensors. Bacterial Antibodies. Antigen-antibody reactions. Immobilized Antibodies. Staphylococcal Protein A.
Conducting polymers. Self assembled monolayers. They have high molecular weights and limited stability, contain essential disulfide bonds and are expensive to produce. The elements of the family that specifically bind to a given target antigen, are often selected in vitro by display techniques: phage display , ribosome display , yeast display or mRNA display. The artificial binding proteins are much smaller than antibodies usually less than amino-acid residues , have a strong stability, lack disulfide bonds and can be expressed in high yield in reducing cellular environments like the bacterial cytoplasm, contrary to antibodies and their derivatives.
The specific binding capabilities and catalytic activity of enzymes make them popular bioreceptors.
Analyte recognition is enabled through several possible mechanisms: 1 the enzyme converting the analyte into a product that is sensor-detectable, 2 detecting enzyme inhibition or activation by the analyte, or 3 monitoring modification of enzyme properties resulting from interaction with the analyte. Notably, since enzymes are not consumed in reactions, the biosensor can easily be used continuously. The catalytic activity of enzymes also allows lower limits of detection compared to common binding techniques. However, the sensor's lifetime is limited by the stability of the enzyme. The association between analyte and receptor then is of reversible nature and next to the couple between both also their free molecules occur in a measurable concentration.
Biosensors that employ nucleic acid interactions can be referred to as genosensors. The recognition process is based on the principle of complementary base pairing , adenine:thymine and cytosine:guanine in DNA. If the target nucleic acid sequence is known, complementary sequences can be synthesized, labeled, and then immobilized on the sensor. The hybridization probes can then base pair with the target sequences, generating an optical signal. The favored transduction principle employed in this type of sensor has been optical detection. It has been proposed that properly optimized integrated optical resonators can be exploited for detecting epigenetic modifications e.
DNA methylation, histone post-translational modifications in body fluids from patients affected by cancer or other diseases.
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Organelles form separate compartments inside cells and usually perform function independently. Different kinds of organelles have various metabolic pathways and contain enzymes to fulfill its function. Commonly used organelles include lysosome, chloroplast and mitochondria. The spatial-temporal distribution pattern of calcium is closed related to ubiquitous signaling pathway. Mitochondria actively participate in the metabolism of calcium ions to control the function and also modulate the calcium related signaling pathways. Experiments have proved that mitochondria have the ability to respond to high calcium concentration generated in the proximity by opening the calcium channel.
Another application of mitochondria is used for detection of water pollution. Detergent compounds' toxicity will damage the cell and subcellular structure including mitochondria. The detergents will cause a swelling effect which could be measured by an absorbance change. Experiment data shows the change rate is proportional to the detergent concentration, providing a high standard for detection accuracy. Cells are often used in bioreceptors because they are sensitive to surrounding environment and they can respond to all kinds of stimulants.
New Materials for the Construction of Electrochemical Biosensors
Cells tend to attach to the surface so they can be easily immobilized. Compared to organelles they remain active for longer period and the reproducibility makes them reusable. They are commonly used to detect global parameter like stress condition, toxicity and organic derivatives. They can also be used to monitor the treatment effect of drugs. One application is to use cells to determine herbicides which are main aquatic contaminant. The algae are continuously cultured to get optimized measurement.
Results show that detection limit of certain herbicide can reach sub-ppb concentration level. Some cells can also be used to monitor the microbial corrosion. The respiration activity is determined by measuring oxygen consumption. There is linear relationship between the current generated and the concentration of sulfuric acid.
The response time is related to the loading of cells and surrounding environments and can be controlled to no more than 5min. Tissues are used for biosensor for the abundance of enzymes existed. Advantages of tissues as biosensors include the following: .
There also exist some disadvantages of tissues, like the lack of specificity due to the interference of other enzymes and longer response time due to transport barrier. The simplest way is to functionalize the surface in order to coat it with the biological elements. Subsequently, the bound biological agent may be for example fixed by Layer by layer depositation of alternatively charged polymer coatings. The most commonly used hydrogel is sol-gel , a glassy silica generated by polymerization of silicate monomers added as tetra alkyl orthosilicates, such as TMOS or TEOS in the presence of the biological elements along with other stabilizing polymers, such as PEG in the case of physical entrapment.
Another group of hydrogels, which set under conditions suitable for cells or protein, are acrylate hydrogel, which polymerize upon radical initiation. One type of radical initiator is a peroxide radical, typically generated by combining a persulfate with TEMED Polyacrylamide gel are also commonly used for protein electrophoresis ,  alternatively light can be used in combination with a photoinitiator, such as DMPA 2,2-dimethoxyphenylacetophenone. Biosensors can be classified by their biotransducer type.
The most common types of biotransducers used in biosensors are:. Electrochemical biosensors are normally based on enzymatic catalysis of a reaction that produces or consumes electrons such enzymes are rightly called redox enzymes. The sensor substrate usually contains three electrodes ; a reference electrode , a working electrode and a counter electrode.
The target analyte is involved in the reaction that takes place on the active electrode surface, and the reaction may cause either electron transfer across the double layer producing a current or can contribute to the double layer potential producing a voltage. We can either measure the current rate of flow of electrons is now proportional to the analyte concentration at a fixed potential or the potential can be measured at zero current this gives a logarithmic response. Note that potential of the working or active electrode is space charge sensitive and this is often used.
Further, the label-free and direct electrical detection of small peptides and proteins is possible by their intrinsic charges using biofunctionalized ion-sensitive field-effect transistors. Another example, the potentiometric biosensor, potential produced at zero current gives a logarithmic response with a high dynamic range. Such biosensors are often made by screen printing the electrode patterns on a plastic substrate, coated with a conducting polymer and then some protein enzyme or antibody is attached. They have only two electrodes and are extremely sensitive and robust.
All biosensors usually involve minimal sample preparation as the biological sensing component is highly selective for the analyte concerned. The signal is produced by electrochemical and physical changes in the conducting polymer layer due to changes occurring at the surface of the sensor. Such changes can be attributed to ionic strength, pH, hydration and redox reactions, the latter due to the enzyme label turning over a substrate.
One such device, based on a 4-electrode electrochemical cell, using a nanoporous alumina membrane, has been shown to detect low concentrations of human alpha thrombin in presence of high background of serum albumin. The use of ion channels has been shown to offer highly sensitive detection of target biological molecules. Capture molecules such as antibodies can be bound to the ion channel so that the binding of the target molecule controls the ion flow through the channel.
This results in a measurable change in the electrical conduction which is proportional to the concentration of the target. An ion channel switch ICS biosensor can be created using gramicidin, a dimeric peptide channel, in a tethered bilayer membrane. Breaking the dimer stops the ionic current through the membrane. The magnitude of the change in electrical signal is greatly increased by separating the membrane from the metal surface using a hydrophilic spacer. Quantitative detection of an extensive class of target species, including proteins, bacteria, drug and toxins has been demonstrated using different membrane and capture configurations.
Electrochemical Sensors, Biosensors and their Biomedical Applications
A reagentless biosensor can monitor a target analyte in a complex biological mixture without additional reagent. Therefore, it can function continuously if immobilized on a solid support. A fluorescent biosensor reacts to the interaction with its target analyte by a change of its fluorescence properties. A Reagentless Fluorescent biosensor RF biosensor can be obtained by integrating a biological receptor, which is directed against the target analyte, and a solvatochromic fluorophore, whose emission properties are sensitive to the nature of its local environment, in a single macromolecule.
The fluorophore transduces the recognition event into a measurable optical signal. The use of extrinsic fluorophores, whose emission properties differ widely from those of the intrinsic fluorophores of proteins, tryptophan and tyrosine, enables one to immediately detect and quantify the analyte in complex biological mixtures. The integration of the fluorophore must be done in a site where it is sensitive to the binding of the analyte without perturbing the affinity of the receptor.
Antibodies and artificial families of Antigen Binding Proteins AgBP are well suited to provide the recognition module of RF biosensors since they can be directed against any antigen see the paragraph on bioreceptors. A general approach to integrate a solvatochromic fluorophore in an AgBP when the atomic structure of the complex with its antigen is known, and thus transform it into a RF biosensor, has been described.
This residue is changed into a cysteine by site-directed mutagenesis. The fluorophore is chemically coupled to the mutant cysteine. When the design is successful, the coupled fluorophore does not prevent the binding of the antigen, this binding shields the fluorophore from the solvent, and it can be detected by a change of fluorescence.
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This strategy is also valid for antibody fragments. However, in the absence of specific structural data, other strategies must be applied. Antibodies and artificial families of AgBPs are constituted by a set of hypervariable or randomized residue positions, located in a unique sub-region of the protein, and supported by a constant polypeptide scaffold.
The residues that form the binding site for a given antigen, are selected among the hypervariable residues. It is possible to transform any AgBP of these families into a RF biosensor, specific of the target antigen, simply by coupling a solvatochromic fluorophore to one of the hypervariable residues that have little or no importance for the interaction with the antigen, after changing this residue into cysteine by mutagenesis. More specifically, the strategy consists in individually changing the residues of the hypervariable positions into cysteine at the genetic level, in chemically coupling a solvatochromic fluorophore with the mutant cysteine, and then in keeping the resulting conjugates that have the highest sensitivity a parameter that involves both affinity and variation of fluorescence signal.
A posteriori studies have shown that the best reagentless fluorescent biosensors are obtained when the fluorophore does not make non-covalent interactions with the surface of the bioreceptor, which would increase the background signal, and when it interacts with a binding pocket at the surface of the target antigen. Piezoelectric sensors utilise crystals which undergo an elastic deformation when an electrical potential is applied to them. An alternating potential A. This frequency is highly dependent on the elastic properties of the crystal, such that if a crystal is coated with a biological recognition element the binding of a large target analyte to a receptor will produce a change in the resonance frequency, which gives a binding signal.
In a mode that uses surface acoustic waves SAW , the sensitivity is greatly increased. This is a specialised application of the quartz crystal microbalance as a biosensor. Electrochemiluminescence ECL is nowadays a leading technique in biosensors. In particular, coreactant ECL operating in buffered aqueous solution in the region of positive potentials oxidative-reduction mechanism definitively boosted ECL for immunoassay, as confirmed by many research applications and, even more, by the presence of important companies which developed commercial hardware for high throughput immunoassays analysis in a market worth billions of dollars each year.
The appropriate placement of biosensors depends on their field of application, which may roughly be divided into biotechnology , agriculture , food technology and biomedicine. In biotechnology, analysis of the chemical composition of cultivation broth can be conducted in-line, on-line, at-line and off-line. As outlined by the US Food and Drug Administration FDA the sample is not removed from the process stream for in-line sensors, while it is diverted from the manufacturing process for on-line measurements.
For at-line sensors the sample may be removed and analyzed in close proximity to the process stream. These techniques are mainly used in agriculture, food technology and biomedicine. In medical applications biosensors are generally categorized as in vitro and in vivo systems.
Biomedical applications of nanobiosensors: the state-of-the-art
An in vitro , biosensor measurement takes place in a test tube, a culture dish, a microtiter plate or elsewhere outside a living organism. The sensor uses a bioreceptor and transducer as outlined above. An example of an in vitro biosensor is an enzyme-conductimetric biosensor for blood glucose monitoring. There is a challenge to create a biosensor that operates by the principle of point-of-care testing , i. A biosensor can be sent directly to the location and a quick and easy test can be used. This book compiles the expert knowledge of many specialists in the construction and use of chemical sensors and biosensors including nitric oxide sensors, glucose sensors, DNA sensors, hydrogen sulfide sensors, oxygen sensors, superoxide sensors, immuno sensors, lab on chip, implatable microsensors, et al.
Emphasis is laid on practical problems, ranging from chemical application to biomedical monitoring and from in vitro to in vivo, from single cell to animal to human measurement. This provides the unique opportunity of exchanging and combining the expertise of otherwise apparently unrelated disciplines of chemistry, biological engineering, and electronic engineering, medical, physiological. Provides user-oriented guidelines for the proper choice and application of new chemical sensors and biosensors Details new methodological advancements related to and correlated with the measurement of interested species in biomedical samples Contains many case studies to illustrate the range of application and importance of the chemical sensors and biosensors.
Chapter 3 Electrochemical glucose biosensors. Chapter 4 New trends in ionselective electrodes. Chapter 5 Recent developments in electrochemical immunoassays and immunosensors.