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Author(s): Rasleen Kaur1, S. Keshavkant2

Email(s): 1rasleen.kaur000@gmail.com, 2skeshavkant@gmail.com

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    1School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur 492 010, India
    2School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur 492 010, India
    *Corresponding Author Email: S. Keshavkant (skeshavkant@gmail.com)

Published In:   Volume - 3,      Issue - 2,     Year - 2021


Cite this article:
Rasleen Kaur and S. Keshavkant (2021) Enzyme Based Biosensor for Onsite Detection of Chromium. NewBioWorld A Journal of Alumni Association of Biotechnology,3(2):1-7.

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NewBioWorld A Journal of Alumni Association of Biotechnology (2021) 3(2):1-7  

REVIEW ARTICLE

Enzyme Based Biosensor for Onsite Detection of Chromium

Rasleen Kaur, S. Keshavkant*

School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur 492 010, India

*Email: skeshavkant@gmail.com


ARTICLE INFORMATION

ABSTRACT

Article history:

Received

 

Received in revised form

 

Accepted

 

 

Environment has own permissible limit of heavy metals (HMs) in its every component (soil, water and air). Excess use of HMs overcomes this limit hence, leading to toxicity affecting the life on Earth. Due to the oxidizing property of the chromium (Cr), one of the HMs, it is widely used for a number of purposes such as manufacturing of stainless steel, in tannery industry, as cleaning agent for glassware, etc. Chromium has two main stable oxidation states; hexavalent (VI) and trivalent (III), out of which earlier is comparatively more toxic than the later form. Solubility of Cr (VI) in ground water had led to the need of designing a sensitive device which can be quite efficient in monitoring of its presence in the environment. In this review, an attempt has been made to collate information, so far available, on the use of microbial system for intake and detoxification of Cr following use of chromate reductase (CR) enzyme.

Keywords: Biosensor; Cromium; Chromate reductase; Enzyme 

Heavy metal

 


Introduction

Metals are constituent of the Earth’s crust with varying content at different places based on the environmental factors. Metallic elements having atomic weight higher than 40.04 (atomic mass of Ca) (Ming-Ho 2005) are considered as heavy metals (HMs). Heavy metals possess range of atomic weights which varies between 63.5 to 200.6 g mol1. Specific gravity defines HMs with value greater than 5 g cm3. Cobalt, copper, iron, manganese, molybdenum, vanadium, strontium and zinc are some of the essential HMs required by living organisms in trace amounts. Non-essential HMs such as cadmium, arsenic, chromium (Cr), lead, mercury and antimony affects surface water system (Oehme and Wolfbeis 1997). Heavy metals are categorized as pollutants adversely affecting the environment. Accumulation of HM in the environment is a serious problem globally. Compared to organic contaminants which are easily degraded in nature, HM contaminants possess non-biodegradable and persistent properties. Heavy metals tend to concentrate in living tissues thus becoming part of food chain causing various health issues in organisms (Kouba et al. 2010; Vinod et al. 2010). Among many environmental problems, HM play negative role and causing soil and water pollution thereby affecting 235 million hectares of arable land worldwide (Bermudez et al. 2012). Enrichment of soil with ions of HMs not only disturbs the biodiversity and productivity but also affects the structure and function of ecosystem (Mayor et al. 2013). Industries and mining sites indiscriminately discharge hazardous and harmful waste containing HMs at elevated concentration thus causing environmental pollution, which is one of the growing concerns all around the world (Ruggaber and Talley 2006). Heavy metals are easily available on the Earth surface due to their natural sources such as natural weathering of the Earth’s crust, mining, soil erosion, etc. (Ming-Ho 2005). Presence of HMs beyond the threshold limits of soil is due to anthropogenic sources such as urban runoff, sewage effluents, industrial discharge, disease control agents applied to plants and a number of other reasons contribute to their abundance leading to toxicity. Heavy metal contamination follows a loop of industry, atmosphere, soil, water, food and human being. Toxicity of HM is a function of concentration (Agency for Toxic Substance and Disease Registry [ATSDR] (2008). High concentration of these leads to carcinogenicity, allergy, inhibition of sensitive enzyme function, etc. (Koropatrick and Leibbrandt 1995).

 

 

Chromium: Transition Element

Among various elements found in Earth crust, Cr exists as 21st most abundantly found metal (Gallios and Vaclavikova 2008). Chromium owns its name from a Greek word “chroma” meaning color. This trace metal gives Ruby and Emerald its red and green color respectively. Chromium holds 24th position in periodic table as member of group VIB d-block element, intermediate to molybdenum and tungsten. It has four naturally occurring non-radioactive isotopes; 50Cr (4.35%), 52Cr (83.80%), 53Cr (9.50%) and 54Cr (2.35%). Atomic mass of Cr is 51.996 g mol-1 and, it has a range of oxidation states from -2 to +6, and is useful for manufacturing of varied chemicals (Shekawat et al. 2015). It exhibits ubiquitous nature and found naturally in water, soil, rocks and air. Chromium concentration in air is <0.01 μg m-3 (ATSDR 2008), in water is 0.2-114.4 μg l-1 (ATSDR 2008) and in soil is 1-1,000 mg kg-1 (Richard and Bourg 1991). Geochemistry of any region is responsible for availability of Cr in the Earth crust. Chromium exists as grey colored solid and melts at high temperature. Its ore has many forms; mineral chromite is the economically extractable form. The annual world production of chromite shows that the South Africa is the leading producer of Cr with India contributing 14% of total output. About 98% of Cr produced from India is contributed by the Sukinda Valley Mines of Odisha alone. The total mean concentration of Cr ranges from 7 to 150 ppm (Jankiewicz and Ptaszynski 2005). However, its content and distribution in the soil mainly depend on the type of mother rock of soil. The presence of additional amount of Cr in the soil is caused by anthropogenic activity. Among various oxidation states; trivalent Cr (III) and hexavalent Cr (VI) species are most prevalent, which differ in their individual physiochemical and toxicological properties (Ishibashi et al. 1990). The Cr(VI) is relatively more water soluble, bioavailable, oxidizing, reactive and toxic than the Cr(III). These properties allows Cr(VI) to easily cross the cell membrane, hence cause oxidative damage to biomolecules (Cervantes et al. 2001). Chromium compounds are considered as mutagenic (Venitt and Levy 1974) and teratogenic (Bauthio 1992) in nature to living systems. Elevated concentration of Cr due to the anthropogenic activities in the environment is major concern. Due to the toxicity of Cr(VI), the U.S. Environmental Protection Agency (EPA) has classified Cr as a Group ‘A’ human carcinogen and is one of the main pollutants. The permissible limit of Cr in the sandy soil up to 15 cm is 2-30 ppm and, loamy soil has 14.8-81.0 ppm (Dudka 1992). The directive of the Ministry of the Environment states that the concentration of Cr in the urban areas, up to depth of 0.3-15 m should be 150-190 ppm. The toxicity and mutagenicity of Cr(VI) are approximately 100 (Beleza et al. 2001) and 1,000 (Czako-Ver et al. 1999) times more than the Cr(III) respectively (Pal et al. 2005).

Anthropogenic Sources

The Earth’s crust contains 0.1-0.3 mg kg-1 of Cr in bound form (Molokwane et al. 2008). Large quantity of Cr is dumped into the environment due to improper handling of Cr containing waste, leakage, accidental release, improper storage conditions and untreated industrial effluent (Zazo et al. 2008). About 35% of Cr(VI) is contributed to the environment by anthropogenic sources. Different oxidation state of Cr has many applications. Elemental Cr(0) is rarely found state which limiting its use. The Cr(II) readily oxidizes into Cr(III) due to unstable nature, hence less useful for industry. The Cr(III) easily adsorbed on soil surface thus has high potential for contaminating aquifers and surface water. The Cr(VI) is most widely used form of Cr in industries. Its compounds are used in the form of chromate and dichromate due to the oxidizing property. Under acidic condition dichromate is used to soak glassware in laboratories (Dhala et al. 2013). Due to the corrosion resistant property, they are popularly used in the steel industry to manufacture stainless steel. In electroplating, they are used as protective coating, construction of alloy products, reactor vessels in nuclear and aircraft, in tannery industry used to soften the animal hides, dye and ceramics syntheses, paint, pigments and wood preservative make use of Cr in large quantity. For leather tanning, 80-90% Cr chemicals are used (Papp 2004). In addition to these, spill and dumping of Cr compounds cause entry into soil thereby environmental problems (Rani and Upadhaya 2013). As a result of excessive use of Cr in manufacturing and industrial sector, about 1,70,000 tons of Cr is discarded as waste, annually, into the environment (Kamaludeen 2003). Combustion of coal and oil releases 1700 metric tons Cr year-1. About 35% of Cr released through human activities is Cr(VI) only.

 

Fig 1. Use of chromium in different industrial sectors.

Chromium Toxicity

The Cr(VI) is categorized under group A human carcinogen because of its mutagenicity (Petrilli and  Flora 1977), carcinogenicity (Gruber and Jennette 1978) and teratogenicity (Gale 1978). Chromium possess high toxicity to plants, animals, microbes, humans and fishes ( ATSDR 2008). Toxicity of Cr(VI) can  results in skin ulcer also known as chrome holes, nasal ulcer, lung dysfunction, respiratory problems, altered kidney functions, altered liver  functioning and immune system (Costa and Klein 2006). The EPA has categorized Cr(III) under Group D. Trivalent Cr being insoluble cannot cross cell membrane (Gonzalez et al. 2003). Moreover, in humans, Cr(III) is an essential trace nutrient that plays an essential role in carbohydrate, lipid, protein and mineral metabolism, and acts as a regulator of insulin activity. Its deficiency can lead to Type II diabetes, heart diseases and nervous system disorders. Therefore, according to the U.S. National Research Council, daily dietary intake of Cr(III) should be 50-200 μg for adults. The daily average intake of Cr(III) from air is less than 0.2 to 0.4 μg, water is 2.0 μg, and  food is 60 μg (ATSDR 1998). Trivalent Cr is required by body in trace amount. Long exposure to Cr(III) may cause adverse effects to humans, animals and plants (Guertin 2004).

Reproductive and Developmental Effects

Chromium (VI) affects the growth of child during pregnancy, while Cr(III) does not possess any effect on  reproductive development.

Chromium Toxicity in Animals

Studies have shown that the exposure of Cr(VI) can lead to  severe developmental effects in mice such as gross abnormalities  and loss of reproductive health like reduction in clutch size, low sperm count, and altered functioning of  seminiferous tubules.

Chromium Toxicity in Plants

The concentration range and regulatory limit of Cr in soil is 0.005-3950 mg kg-1 and 100 mg kg-1 respectively (Salt 1995). Chromium(III) has greater affinity of complex formation with the cell biomolecules leading to their altered functions and affecting central dogma (Nickens et al. 2010). Chromium(VI) affects the plant growth; both morphologically and genetically such as inhibition of electron transport, chromosomal DNA damage, altered transpiration and enzyme activity, decreased nutrient uptake, reduced photosynthesis, seed germination, etc. (Cervantes et al. 2001).

Chromium Removal

A number of physical and chemical treatments such as reduction, precipitation, adsorption, ion exchange, reverse osmosis and electro-dialysis, or disposal through landfill are used to remove Cr from industrial effluent and soil respectively. These methods has disadvantage of incomplete metal removal, high economic cost, high energy and reagent consumption, while also generating secondary waste and polluting environment. Some of the chemical treatments done for Cr removal are feasible only at concentration 1 to 100 mg l-1, and are not effective for lower concentration (Cossich et al. 2002). To overcome the limitation of conventional methods, metal- microbe interaction studies are focused as an alternative solution to clean environment.

Chromium Reduction in Microbes

Bioremediation is an effective and attractive approach for cleaning Cr polluted environment. Microorganisms such as bacteria and fungi play crucial role in bioremediation process. The Cr(VI) reducing microorganisms can be easily isolated from natural and contaminated ecosystems, such group of microbes are named Cr resistant bacteria (CRB) (Schmieman et al. 1998). Gram negative bacteria are more sensitive for Cr(VI) reduction as compared to the Gram positive ones (Coleman 1988). Chromate reduction and, resistance are two independent properties of bacteria. It is not necessary that CRB also participate in Cr reduction. During Cr cycling in soil, oxidation and reduction occurs simultaneously. Detoxification of Cr by reduction from higher oxidation state to lower is easier than the oxidation of Cr(III) to Cr(VI) (Cervantes et al. 2001). The pH of the medium influences the oxidation and reduction of Cr in soil. Oxidative power of Cr is directly proportional to high pH value, while reduction takes place at low pH. Chromium reduction capacity of microorganisms varies strain to strain because of difference in biogeochemical activities and nutrient utilization ability. Chromium(VI) detoxification in CRB is related to plasmid genes. Microbes exhibit several protective systems against metal stress at higher concentration. In bacteria, Cr reduction can either be directly by enzymatic activity or indirectly by producing compound that can reduce Cr(VI). Chromium(VI) has soluble nature due to which it easily diffuses from cell membrane. Within cell, ROS are produced leading to toxicity. Chromium reduction is possible under both aerobic and anaerobic conditions. Some bacteria are capable of both aerobic and anaerobic reduction of Cr.

Biosensors

Abundant release of Cr contaminated industrial waste into the environment causes unavoidable problems. The Cr(VI) released from industries, and high amount of Cr(III) produced due to the natural and induced degradation needs preventive measures. Detection of Cr comes prior to its removal from the environment to avoid its ill effects on life forms. Many different methods such as standard methods, lab based methods, sensors and biosensors are used for detection of Cr in potable water. Till date, methods used for Cr determination includes absorption and emission spectroscopy, inductively coupled plasma mass spectroscopy and chromatographic techniques. These techniques offers high sensitivity, selectivity, reliability and accuracy but has limitations of laboratory testing requiring skilled persons, complicated sample collection, pre-treatment of samples and time taking long protocols (Thompson et al. 1998; Han et al. 2001). Spectroscopic method with the use of diphenylcarbazide chemical is time demanding, complex and not economical. Lab techniques are not used much. Biosensors, the electrochemical methods such as ion-selective electrodes, polarography and other voltametric techniques are more preferred due to the selectivity, sensitivity, simple instrument and shorter response (Han et al. 2001). Biosensors are more precise, rapid and accurate. The biggest property of biosensor is portability i.e. on site monitoring of pollutants. In this review our focus is on detection of Cr with the help of biosensors. International Union of Pure and Applied Chemistry (IUPAC) (namely Physical Chemistry and Analytical Chemistry Divisions) defines biosensor as “a self-contained integrated device that is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is in direct spatial contact with a transduction element” (Pearson et al. 2000). Biosensors are designed with purpose to detect electronic signal directly proportional to the analyte present in the sample. Leland C. Clark Jr. (1962) was the pioneer person in biosensor development. He worked with electrode biosensor to detect glucose.

Design of Biosensor

Biosensor as the name suggests consist of a biological substance that acts as recognition part, which can be enzyme, antibody, DNA, etc. A transducer acts as an interface between biological material and sensing element. The third important component is the amplifying unit to process the data generated through biological interaction and display in some form. Biosensors are categorized based on their bioelement and transducer.

Fig 3.Classification of biosensor based on bioelement used and the sensing property.

Fig4. Working principal of a biosensor.

Cross talk between bioelement and transducer play very important role in the functioning of biosensor. Bioelement is attached on the surface of transducer without loosing its biological functioning and structure loss. Methods such as adsorption of bioelement on transducer surface, crosslinking with the help of functional groups, strong covalent binding immoblization and sol gel entrapments are used to establish connection between bioelement and transducer.  Whatsoever immoblization technique is used, it must fulfill certain criteria like; 

1.      Biological activity of biomolecules to be used in sensing should not get altered during their immoblization over the transducer.

2.      The senstivity of biosensor depends upon how closely the bioelement is bound to the biosensing element. Higher the proximity more is the sensitivity of the biosensor.

3.      The immoblization technique must be durable with biological activity for long run.

4.      Immoblization  method must not interfere with specificity of biosensor for analyte detection (Collings 1997).

5.      The technique should support reproducablity and reuse of same biomaterial (D'Souza 2001).

In biosensor, transducer functions to convert physical, chemical and biological reactions occurring between the bioelement and sensing element into the electrical readable signals (Lowe 2007). On the basis of output signals from transducer different biosensors are designed. Electrochemical biosensor based on electrochemical properties are the most studied since 1960s for glucose oxidase enzyme activity in glucose biosensor (Clark 1962). Various mechanisms involved in the transduction are; electrochemical, electrical, optical, thermal, polarimetric, piezoelectric and surface acoustic wave, optical and field effect transducers, thermal and piezoelectric (Pearson et al. 2000). Response of transducer may vary due to the;

1. Change in hydrogen ion concentration.

2. Reaction involving emission of gases such as ammonia.

3. Electrons being utilized or evolved in reaction.

4. Emission of light.

5. Emission of heat.

6. Absorption or reflectance.

7. Change in mass.

Different electrochemical biosensors are constructed such as impedimetric, potentiometric or amperometric, each with its advantages and disadvantages (Pohanka 2008). Signals generated through interaction between biomolecule and transducer directly relates to the presence and amount of analyte. Of all the electrochemical biosensors, most studied is the amperometric biosensor for Cr sensing in the environment due to easy miniaturization and activity with less volume. From biorecognition side, this review highlights the enzyme based sensing of Cr from contaminated site. Enzyme based biosensors utilize the enzymes active in CRB capable to reduce toxic oxidation state of Cr(VI) to nontoxic state Cr(III). Microbial degradation of environmental HM pollutants is slow as compared to particular enzymes isolated from microbial species (Sutherland et al. 2004). Enzymatic biosensors are more specific in its functioning and devoid of toxic by-products thus not posing any negative impact on the environment. Designing of enzyme based biosensor can use either free enzyme or immobilized enzyme as bioelement. Immoblized enzymes have several advantages over free enzymes, which are listed below (Berezhetskyy et al. 2008);

1.        Quantities of enzymes required to be immobilized on the surface is thousand times lower than the free enzymes for any reaction.

2.        Less interference.

3.        Immobilized enzymes avoid unnecessary pre-incubation.

4.        Rapid analysis of analyte.

5.        Reactivation of enzyme activity is not necessary as in case of reversible inhibition.

Enzymatic biosensors have two modes of actions for sensing of HM ions such as Cr either by inhibition or activation. Sometimes HM ions become integral part of the enzyme structure and acts as their cofactor to enhance the overall functions of enzymes, for example metalloproteins and thus activate the enzyme. Functioning of enzyme biosensor based on inhibition of enzyme activity is due to the interference of metal ions with the thiol or methylthiol groups of amino acids present at the active site of enzymes. Many enzymes such as oxidases and dehydrogenase work on enzyme inhibition (Krawczyk et al. 2000). For Cr determination inhibition based biosensors are developed using L-lactate dehydrogenase, hexokinase and pyruvate kinase. Presence of Cr inhibits the action of these enzymes. Heavy metal ions present in the sample are measured in terms of % Inhibition and it is given like;

% Inhibition = [(Ao – A)/Ao x 100 %]

A: absorbance of substrate obtained before incubation in HM

Ao: absorbance of urea obtained after incubation in HM

Amperometric Biosensor

The amperometric biosensor has two electrodes; working and reference. On applying constant potential, electroactive material is either oxidized or reduced which results in electron flow between two electrodes. The change in current determines the analyte concentration which is related to mass transfer to the electrodes. Caroline et al. (2003) developed first amperometric biosensor using CR enzyme and Cytochrome c3 from anaerobic sulphate reducing bacteria. This biosensor was designed using different immobilization methods. When reduction potential is applied immoblized cytochrome get oxidized and resulting in electron release which is used in Cr reduction. Chromium amperometric biosensor has rapid quantification with sensitivity range 1.8 nA µM-1 and detection limit; 0.20-6.84 mg l-1. With higher Cr concentration, efficiency of sensing was reduced. Aiken et al. (2003) used enzyme inhibition by nitrate reductase for HM detection. Another sensor was designed based on oxidizing ability of Acidothiobacillus fero oxidans using oxygen probe transducer. Bacterial biomass was immobilized on cellulose filter. Bacterial biosensor has very high sensitivity 816 µM l-1 and response time of 51 sec for 2×10-5 concentration Cr2O7. Caroline et al. (2006) quantified Cr in ground water using Cytochrome c3 of D. norvegicum (DSM 1741). Biosensor was assessed under different pH, temperature and ionic strength. Cytochrome c3 was immobilized on glassy carbon electrode with the help of dialysis membrane. The lowest detection limit of Cr(VI) of Cytochrome c3 biosensor was 0.2 mg l-1. Nepomuscene et al. (2007) worked with urease isolated from the Dolichos uniforms. He studied sol gel immobilization method to detect Cr and determined the difference in working of free and immobilized enzyme. Immobilized urease has maximum velocity (Vm) of 3.28 × 10-3 mM min-1 which is very less as compared to free urease 10.62 mM min-1. For restoration of immobilized urease activity, the sensor was soaked in buffer solution containing ethylene diaminetetraacetic acid (EDTA). Maria et al. (2008) developed amperometric biosensor using glucose oxidase enzyme. Electro synthesized poly-o-phenylene diamine was used for immobilizing glucose oxidase. Glucose oxidase amperometric biosensor could easily detect toxic Cr(VI). This study used hydrogen peroxide decomposition by metal ions to estimate enzyme inhibition interference. Ana et al. (2014) used chronoamperometric assay to quantify Cr(III) and Cr(VI) in samples. For Cr(III) detection, tyrosinase isolated from mushroom was immobilized on screen printed carbon electrodes by cross linking with mediator tetrathiafulvalene (TTF). Inhibitory activity of tyrosinase to pyrocatechol was measured. Limit of detection was 2.0±0.2 mM and reproducibility was 5.5%. Quantification of Cr(VI) was done through glucose oxidase isolated from  Aspergillus niger, which was immobilized on modified screen printed electrodes using ferricyanide as mediator. In the presence of Cr, chronoamperometric response was reduced for glucose with limit of detection 90.5±7.6 nM and reproducibility of 6.2%. Aisha et al. (2004) worked with horseradish peroxidase enzyme for Cr detection with the help of hydrogen peroxide. This was the first study utilizing horseradish peroxidase for detection of Cr(III) and Cr(VI) through enzyme inhibition. Enzyme was immobilized directly on carbon film electrode or electropolymerized using gulutaraldehyde with bovine serum albumin. The detection limit was 0.4 and 2.5 μM respectively for Cr(III) and Cr(VI). Amperometric biosensor has two approaches for Cr quantification; the direct approach which utilizes anaerobic Cytochrome c3 showed a linear relation between increase in Cr concentration and current flow. The second approach was indirect quantification following enzyme inhibition activity in the presence and absence of substrate. Biosensor based on indirect approach showed exponential decay of current with Cr concentration. The best amperometric biosensor for Cr detection was urease with the highest detection limit 10×103 to 50×103 μmol l-1.

Conductometric Biosensor

In enzymatic reaction, charged species are either consumed or released which leads to change in ionic strength of sample. Conductometric biosensors are designed with two electrodes dipped in solution. Enzymatic reactions are used to charge neutral species and the change in conductance of medium was measured. Compared to other biosensors, conductometric biosensor offers size miniaturization, cost effectiveness with less power consumption. The Cr(VI) contamination was accessed using thiosulphate oxidizing bacteria (TOB) and, based on thiosulphate oxidation pH, production of sulphate and electrical conductivity were determined. Using thiosulphate as electron donor, biosensor was developed with Cr(VI) detection limit of 100 µg l-1. Many researchers have utilized inhibition activity of sulphur reducing bacteria (SOB) to estimate Cr in sample. Under oxygen rich condition, SOB uses SO as electron donor and oxygen as electron acceptor; and by oxidation reaction produces sulphate and protons which increases the electrical conductivity by increase in protons. Presence of HMs affects oxidation of sulphur to sulphate by SRB. Qambrani et al. (2016) worked for Cr quantification using inhibition activity of SOB, best at 2.0 mg l-1 Cr(VI) with 98% inhibition. Fourou et al. (2016) developed Cr(VI) biosensor based on β-galactosidase inhibition, isolated from Aspergillus oryzoe. The β-galactosidase was immobilized by cross linking with glutaraldehyde on electrochemical transducer. The β-galactosidase was inhibited by the presence of Cr(VI). The limit of detection for this biosensor was 3.12×10-10 M.

Potentiometric Biosensor

Potentiometric biosensor was based on oxidation or reduction potential of an electrochemical reaction. It consists of ion selective electrodes and transistors that convert biological reactions into electrical signals. Nernst equation was used to study relationship between potential difference and analyte in the electrochemical cell.

Ecell =E0cell – RT/nFIn Q

Ecell= Cell potential at zero current

E0cell= Constant potential

R= Universal gas constant

T= Absolute temperature in kelvin

N= Number of charge transferred during electrode reaction

F= Faraday constant

Q= Ratio of anode ion concentration/ Cathode ion concentration

Microbial fuel cell (MFC) as biosensor for real time Cr monitoring was designed by Bingchuan et al. (2001), based on potentiometric principal. It is least utilized type of biosensor.

Resonant Biosensor

Resonant biosensor is based on change in mass of the membrane when analyte molecule is attached to bioelement coupled with acoustic wave transducer. The bioelement in this biosensor is an antibody molecule having affinity for antigen molecule. The change in mass directly changes the resonant frequency which is measured and displayed (Higson et al. 1994).

Optical Biosensor

These biosensors measures the light as electrical signal generated in any reaction. These can be based on optical diffraction or electrochemiluminescence. Optical diffraction biosensors are more efficient to response against minor change in refractive index or thickness when cells get bound to immobilize receptors on transducer. Change in light can be directly correlated to changes in mass number and molecules (Syam et al. 2012).

Thermal Biosensor

Thermal biosensor is based on principal that biological reactions involve liberation or involvement of heat. Such types of biosensors are used in the detection of pesticides and pathogenic bacteria (Syam et al. 2012).

Nanobiosensors

By the integration of nanotechnology with biosensors, nanobiosensors are developed. Nanoparticles such as silver, gold, carbon, etc., are utilized in the sensing of biomolecules with more sensitivity. Such types of biosensors are particularly used in drug delivery system (Rai et al. 2012).

Piezoelectric Biosensor

Piezoelectric (PZ) biosensor has advantage of real-time output, simple operation and economic cost. When the surface of the PZ sensor bounded with a selectively binding substance binds to its counter parts, the crystal mass increases with proportional decrease in resonance frequency of oscillation (Lazcka et al. 2007).

Bioluminescence Sensors

Bioluminescence biosensors are based on enzymes ability to emit photon in the reactions. These sensors are highly useful for bacterial detection. The lux gene in bacteria codes for luciferase enzyme under the control of constitutive or inducible promoter. To detect the concentration of interest of compound the gene is fused with the lux gene resulting in fusion product. By measuring the bioluminensance intensity the concentration of compound can be known (D’Souza 2001).

Microbial Biosensor

Microorganisms provide the environmental condition for reaction to be feasible. They adapt to new conditions by bringing modifications in their genome. Whole cell can be used as bioelement in biosensor either in viable or non-viable form. Metabolism of organic compounds releases carbon dioxide, ammonia and acids as end products which are measured by the transducers. Most of the microbial biosensors are developed to access environmental problems, such as biological oxygen demand estimation based on oxygen depletion rate (Rogers et al. 1999).

Conclusions

In this review, we have summarized various biosensors capable of detecting Cr using different transducer elements. Most widely used electrochemical method for Cr sensing is amperometric biosensor. The CRB has potential to detoxify Cr from toxic higher oxidation state +6 to lower +3 with the help of CR enzyme. Chromium metal exhibit different colors at different oxidation state which can be one of the possible ways for Cr sensing. Chromium detection involving CR as a bio-recognition element in biosensor will possibly be a sensitive technique and will be efficient in detection of Cr, even present in trace amounts, in soil and water samples.

Future Prospects

The use of microbes as a remedy to solve environmental problems is a promising technique. Dumping site of tannery effluent and mining ore site are the mostly Cr polluted sites. Different sophisticated techniques involving complex instrumentation are being used by the researchers to detect Cr. Use of enzyme based biosensors will not only helpful in Cr detection but also detoxify it. Biosensors developed so far has very low limit of detection, therefore; improvements can be made to enhance their detection limits for finer detection. Another improvement in Cr detection following the use of enzyme biosensors is to overcome the detection of background noise which often can lead to false positive detection. Developing such Cr biosensor with very simple operation and quick detection will help us to tackle Cr hazards and its finer sensing.

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