INTRODUCTION:

A biosensor is a sensitive analytical tool which converts biological, signals provided by the analyte into electrical signals. it consists of an immobilized layer of biological material coupled with a transducer. The biological material may be an enzyme or an antibody or an organelle or a hormone or entire cells.¹

Fig-1: Basic diagram of Biosensor

A biosensor is an analytical device which converts a biological response into an electrical signal. The term 'biosensor' is often used to cover sensor devices used in order to determine the concentration of substances and other parameters of biological interest even where they do not utilize a biological system directly.

The biosensor is mainly divided into three sections. Sensor: a sensitive biological element (biological material tissue, microorganisms, organelles cell, antibodies, nucleic acids, etc) receptors, enzymes cell, antibodies, nucleic acids, etc) receptors, enzymes

(i) Sensor: a sensitive biological element biological material (Eg. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc)

(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) third section is the associated electronics, which comprises of signal conditioning circuit (amplifier) processor and a display unit.²

In 1967 Updike and Hicks use the same term enzyme electrode to describe a similar device where again the enzyme glucose oxidase was immobilized in a polyacrylamide gel onto a surface of an oxygen electrode for the rapid and quantitative determination of glucose. Besides amperometry Guilbault and Montalvo in 1969 use glass electrodes coupled with urease to measure urea concentration by potentiometric measurement. Starting from 1970, several others authors start to prove the concept of Biosensors, the coupling of an enzyme and electrochemical sensors. This was at the beginning a Biosensor, a strange research where biological elements were combined with electrochemical sensors.³

Such molecular device that enables sensing of these molecular interactions is called biosensors Another consideration that fascinates lots of researchers regarding exploiting this particular field for exploration is its versatility. It is an interdisciplinary technology and involves the collaborative efforts of engineering, microbiology, physics, chemistry, biology, biotechnology and so on.⁴

Hence the challenge for scientist is to develop or improve some good existing concepts for constructing biosensors applicable on real samples and usable in commercial sphere. The aim of this paper is to provide information on progress done during the period of last 5 years in relation to basic known functional principles of bio recognition elements and transducers in relation to specific biosensors application such as clinical diagnosis, food quality control and environmental screening. New trends including application of non materials are also described.⁵

Analyte:

A substance of interest that needs detection. For instance, glucose is an ‘analyte’ in a biosensor designed to detect glucose.

Bio-receptor:

A molecule that specifically recognises the analyte is known as a bioreceptor. Enzymes, cells, aptamers, deoxyribonucleic acid (DNA) and antibodies are some examples of bioreceptors. The process of signal generation (in the form of light, heat, pH, charge or mass change, etc.) upon interaction of the bioreceptor with the analyte is termed bio-recognition.

Transducer:

The transducer is an element that converts one form of energy into another. In a biosensor the role of the transducer is to convert the bio-recognition event into a measurable signal. This process of energy conversion is known as signalisation. Most transducers produce either optical or electrical signals that are usually proportional to the amount of analyte bio-receptor interactions.

Electronics:

This is the part of a biosensor that processes the trans duced signal and prepares it for display. It consists of complex electronic circuitry that performs signal conditioning such as amplification and conversion of signals from analogue into the digital form. The processed signals are then quantified by the display unit of the biosensor.

Display:

The display consists of a user interpretation system such as the liquid crystal display of a computer or a direct printer that generates numbers or curves understandable by the user. This part often consists of a combination of hardware and software that generates results of the biosensor in a user-friendly manner. The output signal on the display can be numeric, graphic, tabular or an image, depending on the requirements of the end user.⁶

Principle:

The desired biological material (usually a specific enzyme) 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 transducer can convert the product linked changes into electrical signals which can be amplified and measured.¹

Biosensors are analytical devices that employ sensitive biological materials to "recognize" certain molecules and provide information on their presence and amount as a signal convenient for recording and processing. Any biosensor consists of the following three basic components: recognition element, Any biosensor consists of the following three basic components: recognition element, recognition element, which is a bioselective membrane involving various biological structur es; physical transducer; electronic system for signal amplification and recording and for user-friendly data representation.⁷

A biosensor in general utilizes a biological recognition element that senses the presence of analyte (The species to be detected). A biosensor system can be the combination of different entities such as sampling, a biosensor, a system for replenishing information and a data analysis system to implement a biological model which provide information to a human or automated controller. The choice of biological material will depend on a number of factors the specificity, storage, operational and environmental stability and creates a physical or chemical response that is converted by a transducer to a signal.⁸

HISTORY:

Father of the Biosensor Leland C. Clark Jr. (1918–2005) was an American biochemist born in Rochester, New York He is most well known as the inventor of the Clark electrode, a device used for measuring oxygen in blood, water and other liquids, Clark is considered the "Father of Biosensors", and the modern-day glucose sensor used daily by millions of diabetics is based on his research.

Glucose + O2 glusoce oxidase Gluconic acid + H2O2

A negative potential was applied to the platinum cathode for a reductive detection of the oxygen consumption.

O2 + 4H+ + 4 e- →2H2O

The history of glucose enzyme electrodes began in 1962 with the development of the first device by Clark and Lyons of the Cincinnati Children’s Hospital. First glucose enzyme electrode relied on a thin layer of GOx entrapped over an oxygen electrode via a semi permeable dialysis membrane. Measurements were made based on the monitoring of the oxygen consumed by the enzyme-catalyzed reaction.⁹

The first biosensor was invented by Professor Leland C Clark Jnr. and he is known as the father of the biosensor concept. In 1956, Clark published his definitive paper on the oxygen electrode. The concept was illustrated by an experiment in which glucose oxidase was entrapped at a Clark oxygen electrode using dialysis membrane. This biosensor was made from a thin layer of glucose oxidase (GOx) on an oxygen electrode. The amount of glucose was estimated by the reduction in the dissolved oxygen concentration Clark's ideas became commercial reality in 1975 with the successful re-launch (first launch 1973) of the Yellow Springs Instrument Company (Ohio) glucose analyser based on the amperometric detection of hydrogen peroxide. Katz introduced one of the first papers in the field of biosensors with the direct potentiometric determination of urea after urease hydrolysis.¹⁰

Ideal characteristics co biosensor:

There are certain static and dynamic attributes that every biosensor possesses. The optimisation of these properties is reflected on the performance of the biosensor.

Selectivity:

Selectivity is perhaps the most important feature of a biosensor. Selectivity is the ability of a bioreceptor to detect a specific analyte in a sample containing other admixtures and contaminants. The best example of selectivity is depicted by the interaction of an antigen with the antibody.

Reproducibility:

Reproducibility is the ability of the biosensor to generate identical responses for a duplicated experimental set-up. The reproducibility is characterised by the precision and accuracy of the transducer and electronics in a biosensor. Precision is the ability of the sensor to provide alike results every time a sample is measured and accuracy indicates the sensor's capacity to provide a mean value close to the true value when a sample is measured more than once.

Stability:

Stability is the degree of susceptibility to ambient disturbances in and around the biosensing system. These disturbances can cause a drift in the output signals of a biosensor under measurement. This can cause an error in the meas-ured concentration and can affect the precision and accuracy of the biosensor

Sensitivity:

The minimum amount of analyte that can be detected by a biosensor defines its limit of detection (LOD) or sensitivity. In a number of medical and environmental monitoring applications, a biosensor is required to detect analyte concentration of as low as ng/ml or even fg/ml to confirm the presence of traces of analytes in a sample. For instance, a prostate-specific antigen (PSA) concentration of 4 ng/ml in blood is associated with prostate cancer for which doctors suggest biopsy tests. Hence, sensitivity is considered to be an important property of a biosensor.

Linearity:

Linearity is the attribute that shows the accuracy of the measured response (for a set of measurements with different concentrations of analyte) to a straight line, mathematically represented as y=mc, where c is the concentration of the analyte, y is the output signal, and m is the sensitivity of the biosensor. Linearity of the biosensor can be associated with the resolution of the biosensor and range of analyte concentrations under test. ⁹

Working of biosensors:

The electrical signal from the transducer is often low and superimposed upon a relatively high and noisy (i.e. containing a high frequency signal component of an apparently random nature, due to electrical interference or generated within the electronic components of the transducer) baseline. The signal processing normally involves subtracting a 'reference' baseline signal, derived from a similar transducer without any biocatalyst membrane, from the sample signal, amplifying the resultant signal difference and electronically filtering (smoothing) out the unwanted signal noise. The relatively slow nature of the biosensor response considerably eases the problem of electrical noise filtration. The analogue signal produced at this stage may be output directly but is usually converted to a digital signal and passed to a microprocessor stage where the data is processed, manipulated to desired units and output to a display device or data store.²

The biological material is fixed by conventional methods, i.e. physical or membrane entrapment, non-covalent or covalent binding. A contact is made between the fixed biological material and the transducer. The analyte binds to the biological material to form a bound analyte which in turn generated the electronic response that can be measured. Sometimes the analyte is converted to a product which could be associated with the release of heat, gas (oxygen), electrons or hydrogen ions. The transducer then transforms the product associated changes into electrical signals which can be amplified, measured and displayed using the electronic system.¹¹

Eliments of Biosensor:

A biosensor consists of a bio-element and a sensor-element. the bioelement may be an enzyme, antibody, living cells, tissue, etc., and the sensing element may be electric current, electric potential, and so on. A detailed list of different possible bio-elements and sensor-elements is shown in Fig. 1. Different combinations of bio-elements and sensor-elements constitute several types of biosensors to suit a vast pool of applications. The “bio” and the “sensor” elements can be coupled together in one of the four possible ways demonstrated. Membrane Entrapment, Physical Adsorption, Matrix Entrapment, and Covalent Bonding. In the membrane entrapment scheme, a semi permeable membrane separates the analyte and the bioelement, and the sensor is attached to the bioelement. The physical adsorption scheme is dependent on a combination of vander Waals forces, hydrophobic forces, hydrogen bonds, and ionic forces to attach the biomaterial to the surface of the sensor. The porous entrapment scheme is based on forming a porous encapsulation matrix around the biological material that helps in binding it to the sensor. In the case of the covalent bonding the sensor surface is treated as a reactive group to which the biological materials can bind. The typically used bio-element, enzyme is a large protein molecule that acts as a catalyst in chemical reactions, but remains unchanged at the end of reaction.¹²

Fig 2: Eliments of biosensor

Types of Biosensor:

Electrochemical Transduction:

Electrochemical biosensors are typically supplied as electrode setups. They are generally divided in three major categories, depending on the underlying measurable parameter. These are current for ampere-metric biosensors, potential or charge accumulation for potentiometric biosensors and conductive or resistive properties for conductometric or impedimetric biosensors, where both of the latter are usually taken as one category. Amperometric biosensors are typically used to detect small molecules by means of an enzyme, e.g. a peroxidase, catalyzing a redox reaction.

Optical Transduction:

Optical biosensor detectors can generally be divided into labeled and label-free. Labels are e.g. fluorophores or nanostructured materials such as nanoparticles, quantum dots or carbon nanotubes. Detectors using labels typically use the evanescent field outside a waveguide resulting from total reflectance within the waveguide.

Thermal Transduction:

Thermal transduction relies on the principles of calorimetry which requires the measurement of temperature changes. Thermal biosensors typically use thermistors, i.e., temperature dependent resistors, as detector unit. Similar to the term “enzyme electrode” used for the first amperometric biosensor, the first thermal biosensor was called “enzyme thermotor”.

Magnetic Transduction:

Magnetic transduction principles for biosensors usually require magnetic nanoparticles as labels, because the analytes to be detected typically are nonmagnetic. The most commonly used magnetic effect for signal transduction in biosensors is giant magnetoresistance. Magnetoresistance is the change in the resistance of a material due to the application of a magnetic field. If alternating layers of ferromagnetic and non-magnetic metals are used, this change is much greater than expected owing to quantum interferences.

Radioactive Transduction:

The first immunoassay developed by Yalow and Berson in 1959 was a radioimmunoassay (RIA) for detection of insulin . Radioisotopes were not only the first labels used for a long time they also offered one of the most sensitive detection methods for immunoassays. As it is not possible to differentiate radioactivity of bound from free radionuclides, they must be separated. therefore all RIAs follow heterogeneous test formats requiring an immobilization step on a surface.¹³

Resonant Biosensor:

In this type of biosensor, an acoustic wave transducer is coupled with an antibody (bioelement). When the analyte molecule (or antigen) gets attached to the membrane, the mass of the membrane changes. The resulting change in the mass subsequently changes the resonant frequency of the transducer. This frequency change is then measured.

Optical biosensors:

The output transduced signal that is measured is light for this type of biosensor. The biosensor can be made based on optical diffraction or electrochemiluminescence. Optical transducers are particularly attractive for application to direct (label-free) detection of bacteria.

Surface plasmon resonance (SPR) biosensor:

This is an evanescent field based optical sensors using thin gold film for sensing applications. The interaction between analyte flowing over immobilized interactant on gold surface is probed through the detection of reflection minima on photo-detector array sensors. SPR has successfully been applied to the detection of pathogen bacteria by means of immunoreactions.

Piezoelectric biosensors:

Piezoelectric (PZ) biosensor offers a real-time output, simplicity of use and cost effectiveness. The general idea is based on coating the surface of the PZ sensor with a selectively binding substance, for example, antibodies to bacteria, and then placing it in a solution containing bacteria. The bacteria will bind to the antibodies and the mass of the crystal will increase while the resonance frequency of oscillation will decrease proportionally.

Thermal Biosensors:

This type of biosensor is exploiting one of the fundamental properties of biological reactions, namely absorption or production of heat, which in turn changes the temperature of the medium in which the reaction takes place. They are constructed by combining immobilized enzyme molecules with temperature sensors.

Electrochemical Biosensors:

Electrochemical biosensors are mainly used for the detection of hybridized DNA, DNA binding drugs, glucose concentration, etc. Electrochemical biosensors can be classified based on the measuring electrical parameters as:

(i) conductimetric.

(ii) Amperometric.

(iii) Potentiometric.

Compared to optical methods, electrochemistry allows the analyst to work with turbid samples, and the capital cost of equipment is much lower. On the other hand, electrochemical methods present slightly more limited selectivity and sensitivity than their optical counterparts.

Conductimetric Biosensors:

The measured parameter is the electrical conductance/resistance of the solution. When electrochemical reactions produce ions or electrons, the overall conductivity or resistivity of the solution changes. This change is measured and calibrated to a proper scale. Conductance measurements have relatively low sensitivity.

Amperometric Biosensors:

This is perhaps the most common electrochemical detection method used in biosensors. This high sensitivity biosensor can detect electroactive species present in biological test samples. Amperometric biosensors produce a current proportional to the concentration of the substance to be detected. The most common amperometric biosensors use the Clark Oxygen electrode.

Potentiometric Biosensors:

These are the least common of all biosensors, but different strategies may be found nonetheless in this type of sensor the measured parameter is oxidation or reduction potential of an electrochemical reaction.

Bioluminescence sensors:

Recent advances in bioanalytical sensors have led to the utilization of the ability of certain enzymes to emit photons as a byproduct of their reactions. This phenomenon is known as bioluminescence. The potential applications of bioluminescence for bacterial detection were initiated by the development of luciferase reporter phages.

Nucleic Acid-based Biosensors:

A nucleic acid biosensor is an analytical device that integrates an oligonucleotide with a signal transducer. The nucleic acid probe is immobilized on the transducer and acts as the bio-recognition molecule to detect DNA/RNA fragments.

Nano-biosensors:

Nanosensors can be defined as sensors based on nanotechnology. Development of nanobiosensor is one of the most recent advancement in the field of Nanotechnology. The silver and certain other noble metal nanoparticles have many important applications in the field of biolabelling, drug delivery system, filters and also antimicrobial drugs, sensors.¹⁰

Application of Biosensor:

Clinical and Diagnostic Applications:

Among wide range of applications of biosensors, the most important application is in the field of medical diagnostics. The electrochemical variety is used now in clinical biochemistry laboratories for measuring glucose and lactic acid. One of the key features of this is the ability for direct measurement on undiluted blood samples. Consumer self-testing, especially self-monitoring of blood components is another important area of clinical medicine and healthcare to be impacted by commercial biosensors.

1. Biosensors and cancer:

Cancer diagnosis and treatment are of great interest due to the widespread occurrence of the diseases, high death rate, and recurrence after treatment. According to the National Vital Statistics Reports, from 2002 to 2006 the rate of incidence (per 100,000 persons) of cancer in White people was 470.6, in Black people 493.6, in Asians 311.1, indicating that cancer is wide- spread among all races. Cancer can take over 200 distinct forms, including lung, prostate, breast, ovarian, hematologic, skin, and colon cancer, and leukemia, and both environmental factors, and genetic factors are associated with an increased risk of developing cancer.

2. Biosensors and Pathogen detection:

There are three main classes of biological recognition elements which are used in biosensor applications. These are enzymes, antibodies and, nucleic acids. In the detection of pathogenic bacteria, however, enzymes tend to function as labels rather than actual bacterial recognition element.²

3. Medical Instrumentation:

Endoscopes, laser surgery and therpy, angioplasty, temperature sensors (hyperthermia, cardiac monitoring, by thermodilution, tissue thermal damage monitoring), pressure sensor (cardiovascular, intracranial, neurology urodynamics), chemical sensors: blood oximetry, Ph, po_2,pco₂(metabolic and respiratory problems, blood an tissue oxygen content, oxygen, hemoglobin dissociation curve).¹⁴

Industrial Applications:

Along with conventional industrial fermentation producing materials, many new products are being produced by large-scale bacterial and eukaryotes cell culture. The monitoring of these delicate and expensive processes is essential for minimizing the costs of production; specific biosensors can be designed to measure the generation of a fermentation product.

1. Agricultural Industry:

Enzyme biosensors based on the inhibition of cholinesterases have been used to detect traces of organophosphates and carbamates from pesticides. Selective and sensitive microbial sensors for measurement of ammonia and methane have been studied. However, the only commercially available biosensors for wastewater quality control are biological oxygen demand (BOD) analyzers based on micro-organisms like the bacteria Rhodococcus erythropolis immobilized in collagen or polyacrylamide.

2. Food Industry:

Biosensors for the measurement of carbohydrates, alcohols, and acids are commercially available. These instruments are mostly used in quality assurance laboratories or at best, on-line coupled to the processing line through a flow injection analysis system. Their implementation in-line is limited by the need of sterility, frequent calibration, analyte dilution, etc. Potential applications of enzyme based biosensors to food quality control include measurement of amino acids, amines, amides, heterocyclic compounds, carbohydrates, carboxylic acids, gases, cofactors, inorganic ions, alcohols, and phenols. Biosensors can be used in industries such as wine beer, yogurt, and soft drinks producers. ²

Bacterial monitoring:

Among bacteria, common food spoiling related to health hazards include, E. coli strain 0157:H7, Listeria monocytogenes, campylobacter and salmonella. These bacteria are common problems faced by the food industries as they reduce the consumer demands of the food if the food provided by company gets contaminated with these food spoiling biological entities. Salmonella, a rod-shaped bacterium is the major cause of food poisoning, leading to excessive loss of water and salts from the body.

Fungal pathogens detection:

Besides bacteria fungi are also the common cause of food spoilage and causing severe health related problems that may prove to be life threatening in most cases. Fungi that cause food contamination are commonly Botrytis sp., Aspergillus, Colletotrichum and many other fungal species like that. Due to theremarkable specificity, reduced costs and easy and quick monitoring through biosensors, fungal toxins can be also detected using optical SPR biosensors. After their detection, steps to remove them from food products can be taken within the time.

Tissue Engineering:

In tissue engineering, biosensors plays immensely significant role in the applicability of the various applications, such as manufacturing “organ specific onchips” and maintaining the 3-D integrity and configuration of the cell cultures where the fate of tissues/cells is directly associated with the content of small biomolecules (adenosine, glucose, hydrogen peroxides etc.) in the medium. biosensors application with respect to nucleic acids is undeniably significant. Typically, a DNA specified sensor comprises of following three processes;

a) Incorporation of probes over the film of substrate.

b) Contact with the required DNA sequence through analogue bases pairing.

c) Read out in the form of analytically useful signal generated from the chemical signal produced as a result of bases interaction.

Enviromental Monitoring:

Environmental water monitoring is an area in which whole cell biosensors may have substantial advantages for combating the increasing number of pollutants finding their way into the groundwater systems and hence into drinking water. Important targets for pollution biosensors now include anionic pollutants such as nitrates and phosphates. The area of biosensor development is of great importance to military and defense applications such as detection of chemical and biological species used in weapons.

Heavy metals:

Heavy metals, i.e. copper (Cu), cadmium (Cd), mercury (Hg), lead (Pb), zinc (Zn), etc., are recently the major cause of serious environmental pollution problems. These are known for their high toxicity and bioaccumulation attribute in the food chain. A number of bacterial biosensors have been developed for the determination of heavy metals in different environmental samples.

Biochemical oxygen demand (BOD):

Biochemical oxygen demand is one of the significant parameters used in the estimation of the concentration of biodegradable organic pollutants present in a water sample. In routine practice, BOD determination of any sample is a time consuming process.

Nitrogen compounds:

Nitrogen compounds, i.e. nitrates, are widely used by food manufacturing industries as preservatives (increase shelf-life) and chemical fertilizer industries as fertilizer (increase the fertility of the soil. To determine the concentration of nitrogen compounds in water samples, several biosensors have been developed by researchers.

Polychlorinated biphenyls (PCBs):

PCBs are toxic organic compounds, universal environment pollutants; hence several countries banned their production long time ago. Such compounds are highly lypophilic nature, therefore abundant chances of accumulating in the food chain. Numerous biosensors have been developed to detect PCBs in the environment including:

i. Nucleic acid (DNA) based biosensor with chronopotentiometric detection.

ii. Immunosensors with fluorescence.

iii. Electrochemical sensors.

Phenolic compounds:

Phenols and their derivatives, i.e. chlorophenol are distributed commonly in the environment. Such compounds are mainly used in the production of dyes, drugs, plastics, pesticides, detergents, etc. Since, phenolics having high toxicity and possible accumulation in the environment and therefore, their detection and monitoring is essential to protect the environment. Most commonly used biosensors for detection and monitoring of phenolics are:

i. Amperometric biosensor with enzyme (tyrosinase) as bioreceptor for selective detection of phenol in effluent.

ii. A flow-injection chemiluninnescence fiberoptic biosensor detection of chlorophenols.

Organophosphrous (OP) compound:

Organophosphorous compounds are a group of organic chemicals that are commonly used as insecticides, herbicides and pesticides in modern agriculture for controlling pests, weeds and vectors. Pesticides are the substances meant for preventing, destroying or repelling of pest. Pesticides are most widely distributed in water, soil and food. Hormones Increasing human population and intensive farming continuously adding natural and synthetic hormones residues i.e. estradiol, estrone and ethylnilestradiol, in the environment which are adversely affecting human health due to increased incidence of genital cancer and reduction in human sperm counts.¹¹

5. Military and Defense Industry:

In military and defense organizations, portable biosensors can be very useful for detection of toxic gases and the agents of chemical warfare, such as mustard and nerve gas. ‘Bacteria’ are our invisible friends and enemies. Some bacteria aid our digestion, others destroy our poisons. The key to protecting a military unit or community from dangerous bacteria is to detect them before they reach their intended victims. People can then be warned to leave the area or wear protective gears. Bacteria can be detected using biosensors.¹⁵

CONCLUSION:

Application of biosensors in medical diagnosis is extremely successful and has widened to agriculture, environmental and food industries. Enormous research studies are being undertaken by research and development companies and diagnostic centers to develop simple, sensitive and cost effective biosensor technologies.

Biosensor development started around 50 years ago with the development of an “enzyme electrode” for glucose monitoring in blood. Since then, biosensor development is ongoing, with clinical applications still being one of the main driving forces. However, despite the fact that biosensors are promising devices when it comes to fast and easy detection of analytes with low time and effort, they are not yet established in clinical routine, in contrast, for instance, to immunoassay techniques.

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Received on 03.12.2018 Accepted on 08.01.2019

Asian J. Pharm. Res. 2019; 9(1): 27-34.

DOI: 10.5958/2231-5691.2019.00006.6