biosensors;components,types , applications and GMO biosensors

Biosensors are devices that helps to determine the concentration of an analyte in a sample. In this ppt, the definition, components, types, applications and GMO biosensors have been described.

1. 1 Biosensors Submitted to, Dr. Aghil Soorya A Assistant Professor Dept. of Botany St. Teresa’s college Ernakulam Submitted by, Silpa Selvaraj Roll no: 13 II M.Sc. Botany St. Teresa’s college Ernakulam

2. Invented by American biochemist L.L Clark in 1950. Biosensors are analytical devices that measure the concentration of an analyte . In biosensors, a biological material ( enzyme , antibody, whole cell, nucleic acid, hormones ) are used to interact with the analyte . This interaction produces a physical or chemical change which is detected by the transducer and converted to an electrical signal . This signal is interpreted and converted to analyte concentration present in the sample . 2 What are biosensors?

3. Two distinct components; 1. Biological component : enzyme, cell etc. 2. P hysical component : transducer, amplifier etc. The biological component performs two key functions: ( i) It specifically recognizes the analyte . ( ii) I nteracts with it and produce some physical change detectable by the transducer . The biological component is suitably immobilized on to the transducer . (Enzymes are usually immobilized by gluteraldehyde on a porous sheet like lens tissue paper or nylon net fabric ; the enzyme-membrane - affixed to the transducer.) Generally , correct immobilization of enzymes enhances their stability. 3 General features

4. 4 Components of a biosensor The Schematic diagram shown above for the biosensor is mainly divided into three sections. (i) Sensor : a sensitive biological element ( ii) Transducer : it is the detector element (works in a physicochemical way; optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological responsible for the display of the results in a user-friendly way (iii) El ectronics , which comprises of signal conditioning circuit (amplifier), processor and a display unit.

5. 5 The biological component- enzyme, nucleic acid, antibody etc. The analyte must be transported from the solution to the biological component for the reaction- simple diffusion . The biological component interacts with the analyte – produces a physical change close to the transducer; 1. Heat released or absorbed by the reaction. 2. Production of an electrical potential due to changed distribution of electrons. 3 . L ight produced or absorbed during the reaction. 4 . Change in mass of the biological component as a result of the reaction. The transducer detects and measures this change and converts in to an electrical signal . This signal is necessarily small, and is amplified by an amplifier before it is fed into the microprocessor . The signal is then processed and interpreted, and is displayed in suitable units.

6. The desired biological material is immobilized by conventional methods (physical or membrane entrapment, non covalent or covalent binding). This immobilized biological material is in intimate contact with the transducer. The analyte binds to the biological material to form a bound analyte - produces the electronic response that can be measured. In some instances the analyte is converted to a product which may be associated with the release of heat, gas, electrons or hydrogen ions. The transducer measures this interaction and outputs electrical signals which can be amplified and measured. The intensity of the signal output is proportional to the concentration of the analyte . 6 principle

7. Highly specific for the analyte in the presence of other interfering chemicals or foreign materials. The reaction used should be independent of factors like pH, temperature, stirring etc. The response should be linear over a useful range of analyte concentrations. The device should be small and biocompatible in case it is to be used for analysis within the body. Sensitivity : should detect even low concentration of analyte . It should be of low cost, small and easy to use . Assay cost should be lower than conventional tests. Assay should be fast, reliable and repeatable . Reusable . Stability : should give maximum response over a period of time. 7 Properties of a good biosensor

8. 8 Types of biosensors

9. On the basis of the transducer used , biosensors can be; Amperometric biosensors. Optical biosensors. Potentiometric biosensors. Calorimetric biosensors. Piezoelectric biosensors. 9 TYPES OF BIOSENSORS

10. Based on the movement of electrons . Normally a constant voltage passes between the electrodes which can be measured. In an enzymatic reaction that occurs, the product can trans fe r an electron with the electrode surface to be oxidised or reduced. This results in an altered current flow that can be measured. The magnitude of the current is proportional to the substrate concentration , Clark oxygen electrode is the simplest form of amperometric biosensor. Determination of glucose by glucose oxidase is a good example. 10 Amperometric biosensor

11. Blood-glucose biosensor is widely used throughout the world by diabetic patients . It looks like a watch pen and has a single use disposable electrode ( Ag/ AgCl reference electrode and carbon working electrode) with glucose oxidase and a derivative of ferrocene (as a mediator). The electrodes are covered with hydrophilic mesh guaze for even spreading of a blood drop. The disposable test strips, sealed in aluminium foil have a shelf-life of around six months. An amperometric biosensor for assessing the freshness of fish has been developed. The accumulation of ionosine and hypoxanthine in relation to the other nucleotides indicates freshness of fish. A biosensor utilizing immobilized phosphorylase has been developed for this purpose. 11

12. In these biosensors, changes in ionic concentrations are determined by use of ion selective electrodes . pH electrode -commonly used ion-selective electrode, since many enzymatic reactions involve the release or absorption of hydrogen ions . Other electrodes - ammonia-selective and CO selective electrodes . The potential difference obtained between the potentiometric electrode and the reference electrode can be measured-proportional to the concentration of the substrate. lon -selective field effect transistors (ISFET) are the low cost devices that can be used for miniaturization of potentiometric biosensors. A good example is an ISFET biosensor used to monitor intramyocardial pH during open-heart surgery. 12 Potentiometric biosensors

13. 13

14. Optical biosensors are the devices that utilize the principle of optical measurements (absorbance, fluorescence, chemiluminescence etc.). They employ the use of fibre optics and electronic transducers . The word optrode , representing a condensation of the words optical and electrode , is commonly used. A most promising biosensor involving luminescence uses firefly enzyme luciferase for the detection of bacteria in food or clinical samples. The bacteria are specifically lysed to release ATP which is used by the luciferase in the presence of oxygen to produce light – measured by the biosensor. 14 Optic biosensors

15. Optical biosensors for blood glucose estimation . Paper strips impregnated with reagents is used for this purpose. The strips contain glucose oxidase , horse radish peroxidase and a chromogen eg ; toluidine). The following reactions occur. The intensity of the colour of the dye can be measured by using a portable reflectance meter . Colorimetric test strips of cellulose coated with appropriate enzymes and reagents are in use for the estimation of several blood and urine parameters. 15 Luminescent biosensors to detect urinary infections. The microorganisms in the urine, causing urinary tract infections, can be detected by employing luminescent biosensors. T he immobilized (or even free) enzyme namely luciferase is used. The microorganisms, on lysis , release ATP which is used by the enzyme to produce light. The quantity of light output can be measured by electronic devices.

16. Also called as thermometric biosensors or thermal biosensors . Consist of a heat insulated box fitted with heat exchanger ( aluminium cylinder). Analyte solution is passed through a small packed bed column containing the immobilized enzyme. As the substrate enters the bed – gets converted to a product – heat is generated. The difference in temperatures between the substrate and the product is measured by thermistors . Small change in temperatures can be detected. Used for the estimation of serum cholestrol - when cholestrol gets oxidized by the enzyme cholestrol oxidase, heat is generated- measured. Estimations of glucose (glucose oxidase), urea (urease), uric acid ( uricase ) can be done. 16 Calorimetric biosensors

17. Piezoelectric biosensors are based on the principle of acoustics (sound vibrations), hence they are also called as acoustic biosensors. Piezoelectric crystals form the basis of these biosensors. These crystals have positive and negative changes which separate, when subjected to stress. This generates an electric field – piezoelectric effect. Such crystals can be used to assay the mass of analyte that binds to the biological component immobilized on the crystal surface. Determination of analytes using Ag- Ab interaction; Crystal surface is coated with antibodies, which bind to the complementary antigen present in the sample solution. This leads to increased mass, which reduces their vibrational frequency; this change is used to determine the amount of antigen present in the sample. The change in vibration frequency is measured after the crystal has been dried in air. 17 PIEZOELECTRIC BIOSENSORS

18. On the basis of recognition elements used, biosensor can be classified as; Enzymatic Biosensors DNA or RNA biosensors Immunosensors (antibody, antigens, or biomarkers), Whole-cell sensors Microbial biosensors etc . 18

19. Whole cell biosensor : Live or dead microbial cells can be used as the biological component of the biosensor in the place of the usual enzymes. The cells are cheaper, have longer active lifetime and are less sensitive to inhibition, pH and temperature variations than are isolated enzymes. Particularly useful when co-enzyme reactions are necessary. Eg ; potentiometric biosensor for nicotinic acid uses whole cells of Lactobacillus arabinosus as the biological component . 19 Enzyme biosensor: Enzyme are used as recognition elements. Devised on immobilization methods, i.e. adsorption of enzymes by van der Waals forces, ionic bonding or covalent bonding. The commonly used enzymes for this purpose are oxidoreductases , polyphenol oxidases, peroxidases and amino oxidases .

20. Immuno -sensors : Immunological preparation (antibodies) are uses as recognition elements. Established on the fact that antibodies have high affinity towards their respective antigens, Antibodies specifically bind to pathogens or toxins, or interact with components of the host's immune system. DNA biosensor: DNA is used as biorecognition element. ( biorecepetor ) Devised on the property that single-strand nucleic acid molecule is able to recognize and bind to its complementary strand in a sample . 20

21. 21

22. These are the simplest forms of biosensors . They are single use strips of cellulose coated with the appropriate enzyme and suitable reagents. They are dipped into the sample solution, and the presence of the analyte is detected by a change in the colour of the strips , which can be compared with a coloured chart to estimate the approximate amount of the analyte . An example of such test strips is the one used to detect glucose in blood/urine of diabetes patients. The strip contains glucose oxidase, horse-radish peroxidase and a weakly coloured chromogen which is converted into a highly coloured dye on oxidation. 22 Colorimetric test strips

23. 23 APPLICATIONS Biosensors have become very popular in recent years. They are widely used in various fields. Biosensors are small in size and can be easily handled . They are specific and sensitive , and work in a cost-effective manner.

24. Biosensors are successfully used for the quantitative estimation of several biologically important substances in body fluids e.g . glucose, cholesterol, urea . Glucose biosensor - regular monitoring of blood glucose . Blood gas monitoring - critical care and surgical monitoring of patients . Mutagenicity of several chemicals can be determined by using biosensors . Several toxic compounds produced in the body can be detected . A promising biosensor technology for urinary tract infection (UTI) diagnosis along with pathogen identification and antimicrobial susceptibility is under study. 24 Applications in medicine and health

25. Biosensors are being used in the medical field to diagnose infectious diseases. Clinical analysis : Quantitative measurement of cardiac markers . Measurement of metabolites which serve as indicator of prevailing pathogenic parameters or metabolic disturbances: glutamate and acetyl choline which are main causes of degenerative diseases. Hafnium oxide (HfO2): for early stage detection of human interleukin . To monitor susceptibility of chemotherapeutic agents . To monitor respiratory gases e.g ; medical oxygen . In organ replacement procedures like artificial pancreas. 25

26. Biosensors can be used for monitoring of fermentation products and estimation of various ions . T o measure the odour and freshness of foods. Eg ; freshness of stored fish can be detected by ATPase . One pharmaceutical company has developed immobilized cholesterol oxidase system - measurement of cholesterol concentration in foods ( e.g ; butter ). F ood industry- to measure carbohydrates, alcohols and acids , for example, during quality control processes . T o check fermentation during the production of beer, yoghurt and soft drinks . Detecting pathogens in , fresh meat , poultry or fish . 26 Applications in industry

27. Biosensors are very helpful to monitor environmental (air, water) pollution . The concentrations of pesticides and the biological oxygen demand (BOD) can be measured by biosensors . Several environmental pollutants can be evaluated for their mutagenicity by employing biosensors. In military , biosensors have been developed to detect the toxic gases and other chemical agents used during war. 27 Applications in pollution control

28. BOD biosensor Biological oxygen demand (BOD,) is a widely used test for the detection of organic pollution . This test requires five days of incubation . A BOD biosensor using the yeast Trichosporon cutaneum with oxygen probe takes just 15 minutes for detecting organic pollution . Gas biosensors Microbial biosensors for the detection of gases such as sulfur dioxide, methane and carbon dioxide have been developed . Thiobacillus - based biosensor can detect the pollutant SO2 while methane can be detected by immobilized Methalomonas . For carbon dioxide monitoring , a particular strain of Pseudomonas is used. 28

29. 29 Immunoassay biosensors Immunoelectrodes as biosensors are useful for the detection of low concentrations of pollutants . Pesticide specific antibodies can detect the presence of low concentrations of triazines , malathion and carbamates , by employing immunoassays.

30. Biosensors may be used to measure carbohydrates, alcohols, and acids in fermented foods . U sed for quality control processes in food production . The devices, however, need to be kept sterile , frequently calibrated . Enzyme-based biosensors - food quality control to measure amino acids, amides, amines, carbohydrates, heterocyclic compounds, carboxylic acids, gases, inorganic ions, cofactors, alcohols and phenols . U sed in the assessment and analysis of products such as wine, beer and yoghurt . D etection of pathogens in food . Presence of Escherichia coli in vegetables, is a bioindicator of faecal contamination in food. 30 Applications in food industry

31. E. coli has been measured by detecting variation in pH caused by ammonia using potentiometric alternating biosensing systems . Optical biosensor detects the presence of Salmonella and Typhimurium in milk and apple juice. Electrochemical magnetic biosensors are used for detecting salmonella bacteria in milk. Fluorometric biosensor detects and quantifies aflatoxins (agricultural products). Electrochemical antibody/enzyme detects aflatoxins B1 in spices and olive oil, aflatoxin M1 in milk . Electrochemiluminescent aptamer biosensor detects the presence of Ochratoxin A in beer and coffee samples. 31

32. Biosensors may be utilized in a number of agricultural applications, such as; Assessing toxins in soils and crops . D etecting and diagnosing infectious illness in crops . M onitoring of key food process parameters . M easuring animal reproduction . S creening for veterinary drugs . 32 Applications in agriculture

33. QCM (Quartz crystalline Micro balancer) biosensor or Acoustic-based biosensor detects plant pathogens like Ralstonia solanacearum , Pseudomonas syringae pv . and Xanthomonas campestris . High density microelectrode array biosensor detects E. coli bacteria in lettuce . The fabricated biosensor can detect the Cucumber mosaic virus (CMV ). Electrochemical biosensors are used to obtain crop output yields, tests pH or nutrient content and in soil experiments . Electronic nose biosensor ; implemented successfully for the fruit ripeness determination , detection of soil borne pathogens and insect infestations etc . Play a major role in detecting and monitoring organophosphorus levels in the soil and groundwater. 33

34. Enzymatic biosensors most extensively used for the determination of the pesticides . Immunosensors use antibodies or antigens as the specific sensing elements and provide concentration dependent signals. This detects and monitors pesticide residues in rapid speed . Electrochemical acetylcholinesterase biosensors- detect carbamate pesticides in fruits in vegetables . Photosystem 2 -based biosensors are used to detect photosynthetic herbicides . Two bacterial biosensors Enterobacter cloacae and E. coli detects the quantity of nitrate present in the soil. 34

35. Future In the future, developments in nanotechnology will likely integrate with biosensors , building a new era of technologies. Nanomaterials have the potential to enhance the electrochemical, optical, magnetic, and mechanical properties of biosensors . Additionally, they may also help establish single molecule biosensors that will be vital to uncovering vital information about the structure and function of biological mechanisms at the single molecule level. 35 Challenges While biosensor technology has vastly developed over recent years and has been widely adopted in numerous scientific fields, particularly in medicine, they are yet to reach their full potential. In spite of the significant contributions biosensors have made in academic research, there are very few biosensors that have reached commercial success. Generally, this is because it is challenging to translate academic research into commercially viable prototypes .

36. Genetically modified organisms ( GMO) biosensors involve the use of genetically engineered organisms to detect specific substances in the environment. The process generally involves introducing genes into the organism’s DNA that code for proteins capable of recognizing and interacting with the target substance. When the target is present, the modified organisms produce a detectable signal, such as fluorescence or bioluminescence. This signal is then measured to determine the concentration of the target substance. 36 Gmo biosensors

37. Selection of Host Organism : Bacteria, yeast, algae , and even higher organisms like plants can serve as host organisms. Choice depends on the intended application , environmental conditions, and the nature of the target substance. Identification of target : Determine the specific target molecule that the biosensor will detect such as environmental pollutants, pathogens or specific chemicals . Selection of recognition element Identify a recognition element, typically a protein or enzyme that has a high affinity and specificity for the target molecule. This element will be responsible for sensing and interacting with the target . 37 Mechanism

38. Integration of Genes : Introduce the genes encoding the recognition element into the DNA of the host organism. utilizing rDNA technology. Expression of genes The introduced genes are expressed within the host organism, leading to the production of the recognition element. Signal transduction Design the biosensor to generate a measurable signal in response to the interaction between the recognition element and the target molecule. Common signals include fluorescence, bioluminescence or electrochemical signals. 38

39. Response activation ; The engineered organism senses the environment for the target substance. Interaction between the target substance and the engineered genes triggers a response. Signal Output: The activated response produces a detectable signal , such as fluorescence, bioluminescence , or the release of a specific enzyme . Detection System : The detection system includes instruments or methods to capture and quantify the signals produced. Fluorimeters , luminometers , or imaging systems are commonly used depending on the nature of the biosensor . Data interpretation Translate the measured signal into meaningful data, indicating the presence and concentration of the target molecule. 39

40. Applications : Environmental Monitoring : GMO biosensors can be used to detect pollutants, heavy metals, or toxins in air, water, or soil. Healthcare : Detection of specific pathogens in clinical samples. Food Safety : Monitoring for contaminants or spoilage indicators . Advantages : Sensitivity : GMO biosensors can often detect lower concentrations compared to traditional methods. Real-time Monitoring : Continuous and real-time monitoring is possible in some applications. Customization : Genetic engineering allows tailoring biosensors for specific substances. 40

41. Challenges : Ethical Concerns : Concerns about releasing GMOs into the environment. Regulatory Approval : Strict regulations govern the use of GMOs in various applications. Cross-Contamination : Potential unintended effects if GMOs interact with wild-type organisms . Ongoing research focuses on enhancing specificity, stability, and response time of GMO biosensors. Integration with emerging technologies , such as nanotechnology, to improve sensor capabilities. 41

42. Bhalla , N., Jolly, P., Formisano , N., & Estrela, P. (2016). Introduction to biosensors.  Essays in biochemistry ,  60 (1), 1–8. https:// doi.org/10.1042/EBC20150001 Malhotra , S., Verma , A., Tyagi , N., & Kumar, V. (2017). Biosensors: principle, types and applications . IJARIIE. Mehrotra P. (2016). Biosensors and their applications - A review.  Journal of oral biology and craniofacial research ,  6 (2), 153–159. https:// doi.org/10.1016/j.jobcr.2015.12.002 Singh, B.D. (2007). Biotechnology . Kalyani Publishers. https ://ijirem.org/DOC/20-an-overview-on-use-of-biosensor-in-agriculture.pdf . 42 References

43. 43 Thank you