biosensor technology
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Immobilization of biomolecules such as enzymes and cells provides a basis for their reuse in biosensors. There are several methods for immobilization including physical entrapment, microencapsulation, adsorption, and covalent binding. Biosensors consist of a bioreceptor such as an enzyme, antibody, or nucleic acid and a transducer that converts the biorecognition event into a measurable signal. Common types of biosensors include enzyme biosensors that detect the products of enzyme-substrate reactions, immunosensors that detect antigen-antibody binding, and DNA/RNA biosensors that detect nucleic acid hybridization. Biosensors have applications in medical diagnostics, environmental analysis, and food quality control.
biosensor technology
- 1. 1 of 7 Biosensors technology Immobilization of Biomolecules may be physically in a solid support through hydrophobic or ionic interactions or covalently immobilized by attachment to activated surface groups. The immobilization of enzymes and cells provides a basis for the re-use of enzymes and cells. Immobilized enzymes: an isolated or purified enzyme confined or localized in a defined volume of space. Immobilized cells: a high density of cells physically confined on a solid phase or in pellets or clumps and in which cell movement is restricted for the period of their use as biological agents Immobilization Methods • Physical entrapment - viscous aqueous solution trapped by membrane permeable to analyte. Membranes such as cellophane, cellulose acetate, PVA, polyurethane Entrapment Gels such as agarose, gelatin, polyacrylamide, poly(N-methyl pyrrolidone) • Microencapsulation: inside liposomes, or absorbed in fine carbon particles that are incorporated in a gel or membrane • Adsorption: direct adsorption onto membrane or transducer; can also be adsorbed onto pre-adsorbed proteins, e.g., albumin; avidin (via biotin linker) • Covalent binding (via –COOH, -NH2, -OH chemistries) or crosslinking (ex., via glutaraldehyde) to transducer or membrane surface Advantages of Immobilized enzymes and cells • Immobilized enzymes are more stable over broad ranges of pH and temperature. • Greater ease of new applications for industrial and medical purposes. • Immobilized enzymes permit the use of enzymes from organisms which would not normally be regarded as safe (i.e. non-GRAS).
- 2. 2 of 7 Biosensors: = bioreceptor + transducer. The bioreceptor is a biomolecule that recognizes the target analyte, and the transducer converts the recognition event into a measurable signal. IT selectively and quantitatively detects the presence of specific compounds in a given external environment. Biosensor consists of: • Bioreceptor (eg. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc), a biologically derived material or biomimic) the sensitive elements can be created by biological engineering. • The transducer (works in a physicochemical way; optical, piezoelectric, electrochemical, etc.) should be capable of converting the biorecognition event into a measurable signal. Typically, this is done by measuring the change that occurs in the bioreceptor reaction. • Associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. This sometimes accounts for the most expensive part of the sensor device, however it is possible to generate a user friendly display that includes transducer and sensitive element Biosensor Characteristics. (1) Sensitivity is the response of the sensor to per unit change in analyte concentration. )2( Selectivity is the ability of the sensor to respond only to the target analyte. That is, lack of response to other interfering chemicals is the desired feature. )3( Range is the concentration range over which the sensitivity of the sensor is good. Sometimes this is called dynamic range or linearity. (4) Response time is the time required for the sensor to indicate 63% of its final response due to a step change in analyte concentration. (5) Reproducibility is the accuracy with which the
- 3. 3 of 7 sensor’s output can be obtained. (6) Detection limit is the lowest concentration of the analyte to which there is a measurable response. (7) Life time is the time period over which the sensor can be used without significant deterioration in performance characteristics. )8 Stability characterizes the change in its baseline or sensitivity over a fixed period of time. Biosensors can be classified either by the type of biological signaling mechanism they utilize or by the type of signal transduction they employ. o Enzyme based sensor: Enzymes are very efficient biocatalysts, which have the ability to specifically recognize their substrates and to catalyze their transformation. High specificity of enzyme– substrate interactions and the usually high turnover rates of biocatalysts are the origin of sensitive and specific enzyme-based biosensor devices. Ideally enzyme catalytic action can be influence by several factors such as the concentration of the substrate, temperature, presence of competitive and non- competitive inhibitor. Glucose oxidase (GOD) and horseradish peroxidase (HRP) are the most widely used enzyme based biosensor. enzyme based biosensor can be used to detect cholesterol, food safety and environmental monitoring, heavy metals and also Pesticides. o Immunosensors : An antibodies based biosensor was applied to the possibility of immuno-diagnosis. Since then, there has been vigorous effort made to develop immunosensor which composed of Antigen/antibody as bioreceptor as a tool for clinical diagnostics.An antibody is ‘Y’ shaped immunoglobin (Ig) that is made up of two heavy chains (H) and two light chains (L). However some of human antibodies form dimeric or pentameric structure by utilizing disulphide bonds and an extra protein called the joining or J- chain. Each of the chain has a constant and variable part. The variable part is specific to the antigen that is bind with corresponding antigen which is highly specific and selective. Hence, an immunosensor which composed of antigen as bioreceptor utilizes the ability of antibody to bind with corresponding antigen which is highly specific, stable, and versatile. For bacteria and pathogen detection has gained a great deal of attention due to its application in the point of care measurement (POC).eg. HCG Pregnancy test
- 4. 4 of 7 o DNA/Nucleic acid sensor: The highly specific affinity binding’s reaction between two single strand DNA (ssDNA) chains to form double stranded DNA (dsDNA) is utilized in nucleic acids based biosensor which appoint the nucleic acids as biological recognition element. This biosensor working principal is based on recognition of the complementary strand by ssDNA to form stable hydrogen bond between two nucleic acids to become dsDNA. In order to achieve this, an immobilized ssDNA is used as probe in bioreceptor eptor which the base sequence is complementary to the target of interest. Exposure of target to the probe which results in hybridization of complementary ssDNA to form dsDNA will result in producing biochemical reaction that allows transducer amplified the signal into electrical one. Subsequently the present of some linker such as thiol or biotin is needed in the effort to immobilize the ssDNA onto the sensing surface.. An important property of DNA is that the nucleic acid ligands can be denatured to reverse binding and the regenerated by controlling buffer ion concentration. The nucleic acid biological recognition layer which incorporates with transducer is easily synthesizable, highly specific and reusable after thermal melting o the DNA duplex. In addition, this biosensor possesses a remarkable specificity to provide analytical tools that can measure the presence of a single molecule species in a complex mixture. DNA based biosensor has potential application in clinical diagnostic for virus and disease detection. o Cell based sensor: use living cell as the biospecific sensing element and are based on the ability of living cell to detect the intracellular and extracellular microenvironment condition, physiological parameter and produces response through the interaction between stimulus and cell. Microorganisms such as bacteria and fungi can be used as biosensors to detect specific molecules or the overall ‘‘state’’ of the surrounding environment. Furthermore, proteins that are present in cells can also be used as bioreceptors for the detection of specific analyte. The detection limit of this biosensor is mainly determined by the natural environmental conditions in which the cell can stay alive for long period where need the control the physical and chemical parameter of environment. However the major limitation with cell based biosensor are the stability of the cell, which depends on various conditions such as the sterilization, lifetime, biocompatibility and etc. The cell based biosensor are less sensitive to inhibition by solutes and are more tolerant of suboptimal pH and temperature values than enzyme based biosensor. Cell based sensor have become an emerging tools for medical diagnostics (i.e. such as disease detection), environmental analysis, food quality control, chemical-pharmaceutical industry and drugs detection. o Biomimetic sensor: A biomimetic biosensor is an artificial or synthetic sensor that mimics the function of a natural biosensor. These can include aptasensors, where aptasensors use aptamers as the biocomponent. Aptamers were described as artificial nucleic acid ligands. Aptamers were thus chemically related to nucleic acid probes, but behaved more like antibody and showing surprising versatility compared to other bio- recognition components. Aptamer are synthetic strands of nucleic acid that can be designed to recognize amino acids, oligosaccharides, peptides, and proteins. An aptamer has few advantages over antibody based biosensor such as high binding efficiency, avoiding the use of animal (i.e reduced ethical problem), smaller and less complex, and etc. However, common challenge facing aptasensor is that they inherent the properties of nucleic acids such as structural pleomorphic and chemical simplicity which reduced the assay efficiency and also increase its production cost. Subsequently, some effort has been directed towards characterization and optimization
- 5. 5 of 7 of aptamer to overcome this limitation. Aptamer properties such as their high specificity, small size, modification and immobilization versatility, regenerability or conformational change induced by the target binding have been successfully exploited to optimize a variety of bio-sensing formats. Aptamer based biosensor has been widely used in various application. biomimetics sensor and aptasensor for clinical application. This including clinical diagnostics to detect pathogen, virus and infectious disease. Transducers o Electrochemical: translate a chemical event to an electrical event by measuring current passed (amperometric = most common), potential change between electrodes, etc. o Photochemical: translate chemical event to a photochemical event, measure light intensity and wavelength (λ) • Colorimetric: measure absorption intensity. • Fluorescence Example 1: DNA microarrays– fluorophores selectively bound to detected molecule via avidin-biotin complex; commercialized by Affymetrix. o Piezoelectric: translate a mass change from a chemical adsorption event to electrical signal Example: Quartz Crystal Microbalance Applications of Biosensors o Glucose monitoring in diabetes patients and other medical health related targets o Detection of pathogens o Routine analytical measurement of folic acid, biotin, vitamin B12 and pantothenic acid as an alternative to microbiological assay Glucose biosensor: knowing the concentration of glucose is critical to proper care of diabetics. Glucose oxidase catalyzes the following reaction: glucose + O 2 → δ - gluconolactone + H 2 O 2 The glucose biosensor consists of a thin layer of glucose oxidase attached to the bottom of an oxygen electrode. The electrode detects oxygen released by the enzyme reaction. The current generated provides a measure of the glucose concentration. A potential of about 0.6 volts is applied between the central positive platinum electrode and the surrounding negative silver/silver chloride electrode. The electrolyte solution is saturated potassium chloride. The negative electrode (cathode) is covered by a thin Teflon membrane, which allows oxygen to diffuse through but keeps out other molecules that might react. Three potential measurement routes: 1. pH change (acid production) 2. O2 consumption (fluorophore monitor) 3. H2O2 production (electrochemical) There is growing interest today in using biosensors to detect toxins, viruses, and perhaps other possible biowarfare agents.
- 6. 6 of 7 BOD biosensor- Biological oxygen demand (BOD) is widely used as a test to detect the levels of organic pollution. This requires five days of incubation but a BOD biosensor using the yeast Trichosporon cutaneum with oxygen probe takes only 15 minutes to detect organic pollution Biochips can be defined as ‘microelectronic-inspired devices that are used for delivery, processing, analysis ،or detection of biological molecules and species’ .These devices are used to detect cells ،microorganisms, viruses, proteins, DNA and related nucleic acids, and small molecules of biochemical importance and interest. Microarrays technology is transforming laboratory research because it allows us to analyze tens of thousands of samples simultaneously. Researchers currently use microarray technology to study gene structure and function. Thousands of DNA or protein molecules are arrayed on glass slides to create DNA chips and protein chips, respectively. Recent developments in microarray technology use customized beads in place of glass slides. DNA Microarrays: DNA microarrays are used to Detect mutations in disease-related genes. Monitor gene activity. Diagnose infectious diseases and identify the best antibiotic treatment. Protein Microarrays: The structures and functions of proteins are much more complicated than that of DNA, and proteins are less stable than DNA. Each cell type contains thousands of different proteins, some of which are unique to that cell’s job. In addition, a cell’s protein profile varies with its health, age, and current and past environmental conditions. Protein microarrays will be used to: Discover protein biomarkers that indicate disease stages. Assess potential efficacy and toxicity of drugs before clinical trials. Study the relationship between protein structure and function.
- 7. 7 of 7 Tissue Microarrays which allow the analysis of thousands of tissue samples on a single glass slide are being used to detect protein profiles in healthy and diseased tissues and validate potential drug targets. Brain tissue samples arrayed on slides with electrodes allow researchers to measure the electrical activity of nerve cells exposed to certain drugs. Whole Cell Microarrays circumvent the problem of protein stability in protein microarrays and permit a more accurate analysis of protein interactions within a cell. Small Molecule Microarrays allow pharmaceutical companies to screen tens of thousands of potential drug candidates simultaneously. Future Directions Multianalyte capability (proteins, biowarfare agents, pathogens, etc.) Integration/Miniaturization (microfluidic “lab on a chip” devices) Implantable Devices Living cells/tissues as biological element