18 Biosensors: An Introduction

Dr Hardeep Kaur

Biosensors: An Introduction

1.What are biosensors

2.Components of biosensor

2.1 Bioreceptors

2.1.1 Enzyme

2.1.2 DNA

2.1.3 Antibodies

2.1.4 Whole cells

2.2 Biotransducers

2.2.1 Electrochemical

2.2.2 Optical

2.2.3 Piezoelectric

2.3 Biosensor construction and characteristics

2.3.1 Immobilization techniques

2.3.2 Biosensor performance attributes: Characteristics

2.3.3 Biosensor Characterization techniques

1 What are biosensors

A biosensor is an analytical device that can convert a biological signal in a measurable electrical or electronic signal. IUPAC recently proposed a very stringent definition of a biosensor , “A biosensor is a self-contained integrated device which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (bioreceptor) which is in direct spatial contact with a transducer. This clearly indicates that a biosensor composed of two main components, a bioreceptor and a transducer. The bioreceptor interact with the analyte (target compound such as toxins, heavy metals, pesticides etc) and produces a biological signal which cannot be perceived by us. So, transducer is incorporated in close proximity with the bioreceptor to convert that biological signal into an electrical signal which is amplified and then displayed by the signal processing unit (Figure 1).

Biosensors are an alternative to big analytical instruments used for detection of various environmental pollutants, food adulterants and clinically important compounds. These instruments include atomic absorption spectrophotometer (AAS), inductively coupled plasma mass spectrometer etc. Although very specific and sensitive to analytes these huge instruments suffer various limitations. The limitations of these conventional techniques and advantages of biosensors over them are listed in table 1. The benefits of high selectivity, sensitivity and fast response time provided by biosensors make them the desirable devices that can be used by common man. A simplest example of a biosensor is a glucometer which people use at home and in hospitals to check blood glucose level. A clear comparison of size and complex nature of AAS and ICP-MS (used for detection of heavy metals in samples) with a glucometer is shown in figure 2. This indicates that use of biosensors is extremely simple and is not a laboratory bound method of sample analysis. Biosensors are also valuable when cost per sample is compared.

2 Components of a Biosensor

In order to understand the functioning of a biosensor, it is necessary to know about the constituents of a biosensor. As already mentioned biosensor consist of mainly two components, a bioreceptor and a transducer. This section is dedicated to explain these in details.

2.1 Bioreceptors

A bioreceptor is a biological entity which can efficiently sense a target and produce a convertible biological signal. The signal produced is usually selective to and proportional to target concentration. According to this any biological molecule such as enzymes, proteins, DNA, whole cells, antibodies etc may act as bioreceptors.

2.1.1 Enzymes

Enzymes are biocatalyst which accelerates the product formation in a chemical or biochemical reaction. Their specificity to particular substrate and cofactors makes them suitable bioreceptors for biosensor development. More over the enzymes capability to a catalyse a number of reactions and suitability to be used with different transducers facilitate their candidature as bioreceptors. Enzyme based analyte recognition could be done through various mechanisms:

1. Production of detectable product by the enzyme catalysed reaction.
2. Detection of analyte dependent activation or inhibition of enzyme activity.
3. Change in enzyme characteristics due to enzyme- analyte interaction.

They are the most exploited bioreceptors and perhaps the first one used in the development of a biosensor. A landmark in biosensor field was the development of Clark oxygen electrode which was fabricated to sense blood glucose levels in diabetic patients. The enzyme glucose oxidase (GOD) was immobilized at the tip of an oxygen permeable electrode that could sense the decrease in oxygen concentration which was proportional to glucose concentration as per the following reaction. The GOD immobilized catalysed the oxidation of glucose by molecular oxygen and caused decrease in oxygen concentration which was sensed by the electrode. The oxygen consumption was detected by the electrode as change in potential.

This is the working principle of first enzyme electrode. Later on similar principal has been used to construct commercial glucometer which also contains GOD as bioreceptor (figure 3).

A number of enzymes have been used to construct biosensors for a wide range of analytes as per table 2. Mostly enzyme based biosensors are constructed on inhibition phenomenon. Whereby the presence of pesticides and heavy metals inhibit the enzyme’s activity and declines rate of product formation.

2.1.2 DNA

DNA (Dideoxy Nucleic Acid) known as genetic material could also act as bioreceptor against complementary sequences and help in disease diagnosis based on hybridization phenomenon. The hybridization of complementary sequence with the bioreceptor DNA on transducer platform produces a detectable signal that could indicates the presence of clinical abnormality.

An advancement in DNA based biosensors is the use of aptamers or DNAzymes that are small nucleotide sequences (20- 30bp) capable of binding to specific compounds. In this field lead ions specific DNAzymes and cancer proteins (biomarkers) specific aptamers are most noteworthy. The details of heavy metal monitoring through DNAzyme approach has been dicussed in further chapters.

2.1.3 Antibodies

Antibodies are immune elements produced inside our body in response to foreign entities called antigens. The biosensors containing antibodies as bioreceptors are often called immunosensor. The antibodies could be purified as monoclonal or polyclonal antibodies and used as bioreceptors for the detection of specific antigen. The formation of antigen-antibody complex is linked to the production of a physicochemical reaction through a transducer and is proportional to the antigen concentration figure 4. This way these immunosensor provide valuable platform for clinical diagnosis of abnormalities and detection of toxins or pathogens.

2.1.4. Whole cells

The whole cells find their application as bioreceptors as they may furnish functional information (information about the effect of a stimulus on a living system) or analytical information (qualitative and quantitative). The intracellular and unstable nature of enzymes and their expensive and lengthy purification methods indicates whole cells as better option. Also the detection schemes that need a sequential use of enzymes of a cell would be more benefitted by the use of whole cells rather than individual purified enzymes. Whole cells also provide the opportunity of producing recombinants that may react to specific analytes such as heavy metals. The heavy metal induced activation of operons lead to generation of either coloured product or fluorescence. Whole cells have been profoundly used for environmental monitoring of pesticides, heavy metals and biological oxygen demand (BOD). These applications have been discussed in details in further chapters.

2.2 Biotransducers

Biotransducer is a physicochemical assembly that is in close contact with the bioreceptor and help in signal conversion into a measurable format. Every biochemical reaction gives rise to a kind of signal which we cannot perceive directly such as, gain or loss of electrons or protons, emergence or absorption of light or fluorescence, change in thermal activity etc. these changes are converted into electrical or electronic signal by the biotransducers. Depending upon the kind of signal perceived biotransducers are classified as per scheme 1

2.2.1 Electrochemical transducers

These are the most common type of transducers used in the development of biosensors because they may perceive a wide range of signals. All detections are based on the simple electrochemistry rules and generally refer change in ions, current or conducvity profiles of the sample. As per their name we can easily get the mode of their working.

Potentiometric biotransducer: This category includes ion selective electrodes which convert biological signal into electrical signal. The simplest example could be a pH meter with an immobilized biocomponent (enzyme) at the tip which causes change in hydrogen ions (H+) concentration of the sample. The pH meter senses change in H+ ion concentration and hence display the action of enzyme as an electric signal. Similar electrodes containing membranes at the tip selective to specific ions such as ammonium ions (NH4+), or gases such as CO2, NH3 or H2S are used for development of ion or gas biosensors. The response of an ion selective electrode is given by Nernst Equation:

E =  Eo + RT/zF ln [I]

Where,

E = measured potential (volts)

Eo = Characteristic constant for the ion selective electrode R = Gas constant

T = absolute temperature (K)

Z = signed ionic charge

F= Faraday constant

I = concentration of ionic species

The ion concentration is related logarithmically to the potential that means that there will be an increase in the electrical potential of 59 mv for every tenfold increase in the ion concentration at 25 º C.

Amperometric biotransducers: As the name suggest, they sense the change in ampere or current (electron flow) due to the biochemical reaction which mainly includes oxidation or reduction reactions. This analysis needs a three electrode assembly which consist of a working electrode, reference electrode and an auxiliary (counter) electrode. The biochemical reaction pertaining to analyte- bioreceptor interaction takes place at the surface of working electrode and reference electrodes provide a stable potential during the analysis. The current flows between the working and the counter electrode and change in current is measured as a function of applied voltage. An electrolyte as potassium chloride (KCl) is used for efficient electron transfer. The Clark electrode is an example of amperometric biosensor. Now days the electrochemical cell has been transformed into small chips which contain the three electrode assembly over Teflon support and a single drop of sample is required for analysis. The strips used in the glucometer are the example and they are called as screen printed electrodes (figure 5).

Conductometric biotransducer: These sensors meaure change in electrical conductivity of the sample. The interaction of bioreceptor and analyte may cause change in ionic species concentration. This in turn changes the electrical conductivity or electron flow in the system. A current is applied between the electrodes and the change in current flow as a function of change in ionic species is measured.

2.2.2 Optical Transducers

Optical as the name suggests, sense change in optical properties of the sample as function of analyte-biocomponent interaction. It includes all the light mediated biochemical reactions which either produces change in phase, amplitude or frequency of input light or coloured products from colourless compounds or change in fluorescence, luminescence.

Fluorescence resonance energy transfer (FRET): FRET is a light emission phenomenon observed between two fluorophores. Fluorophores are the chemical compounds that absorb a particular wavelength of light, undergo excitation and then emit light at a higher wavelength. To observe FRET, the fluorophores should be selected wisely so as the emission wavelength of the donor should overlap the excitation wavelength of the ac- ceptor. Certain compounds that act as acceptors decrease the fluorescence intensity and are called quenchers.

Surface Plasmon resonance (SPR): SPR biotransducers provide an opportunity of real time sensing of bimolecular interaction in a rapid, sensitive and label-freeway. It works on simple principle of change in refractive index of the sensing medium on gold surface generally due to bimolecular interactions that result in change in reflected light or resonance angle (figure 6). SPR have found wide application in immunosensors where the antigen-antibody interactions have been measured through this technique.

(http://spie.org/newsroom/0882-anisotropic-surface-plasmon–resonance-imaging-biosensor)

2.2.3. Piezoelectric

Piezoelectric transducers measure change in mass over the sensing platform due to analyte-bioreceptor interaction. A piezoelectric quartz crystal is resonated at a frequency and change in mass of the biolayer present on the crystal is measured as a function of change in resonance frequency. The interaction of bioreceptor with its anlyte or antigen – antibody interaction could be assessed through this method. More will be the mass on the crystal more will be the shift in frequency as illustrated in figure 7. The change in mass after target binding is directly associated with the target concentration according to the following equations:

2.3 Biosensor construction and characterization

Biosensor construction is a step wise process and need consideration of every component to get successful detection of an analyte. The first step in the process is the selection of analyte specific bioreceptor and its compatible transducer. As evident from the first chapter, a biosensor is destined to convert a biological signal into an electronic signal which predicts the analyte concentration in the sample through interaction with bioreceptor. So, it makes it entirely essential that the bioreceptor should be in an intimate contact with the transducer to transfer the signal immediately without any loss. For this the bioreceptors are immobilized on or near the transducer assembly. This chapter will discuss the various immobilization techniques used to capture bioreceptors close to transducer. After the development of a biosensor, the most important considerations are the performance attributes such as linear range of detection, limit of detection, response time etc., which will be discussed here in details along with the techniques used for biosensor characterization.

2.3.1. Immobilization techniques

Immobilization of bioreceptor is helpful in systemic reuse of biosensor and to get reliable results without variation in bioreceptor concentration. In addition, immobilized system provides a number of advantages over free system such as higher storage stability, stable fabrication methods and favourable environment for bioreceptor activity. The attachment of bioreceptor to the transducer should be done in a way that the characteristics of the bioreceptor important for analyte detection are not lost. In case of enzymes it is entirely necessary to use the immobilization technique that does not affect the active site of enzymes. The main techniques of immobilization used in biosensor construction are: physical adsorption, covalent bonding, crosslinking and encapsulation (figure 8).

Physical methods of immobilization

Adsorption and encapsulation comes under physical methods of immobilization that involve non-covalent interactions between the bioreceptor and the transducer surface. Such interactions include electrostatic, hydrophobic and vander waals forces (figure 9a). Direct immobilization of proteins and enzymes on electrode surface without the use of any cross- linker is the example of physical adsorption. Adsorption could be done with polymer matrix with enhances the enzyme or protein retaining efficiency. But, still, direct adsorption always cause 70 – 80% leakage or percolation of immobilized protein/enzyme. Entrapement or encapsulation of enzymes or proteins is an alternative to prevent loss caused by leakage (figure 9b). Sandwich entrapement of enzymes in sol-gel matric is an efficient method of enzyme immobilization and has been widely used. Layer- by- layer (LBL) assembly of enzymes with sol-gel matrix provide valuable benefits as:

• Entrapement of large amount of enzyme/proteins
• Chemically inactive and optically clear matrix
• No covalent modification of the enzyme
• Makes enzymes thermally stable

LBL assembly of positively and negatively charged reagents is also an alternative foe physical adsorption of proteins on solid surface.

Chemical methods of immobilization

This method includes formation of covalent bond between the bioreceptor and the solid support. The free carboxyl (COOH) and amine groups (NH2) on the proteins are utilized for covalent bond formation with similar groups on the support surface. The solid support is functionalised with chemicals which impart –COOH and –NH2 gps on their surface. An example is the use of cysteamine which makes a thio (SH) group mediated bond with gold surface of an electrode and provide the amine group for further attachment of proteins or antibodies (figure 10). The covalent nature if this kind of bonding makes the immobilization stronger and stable for repeated use. The protein leakage is also reduced to a great extent, but it can affect the active site configuration of the enzymes. So, it is necessary that only the peripheral groups of enzymes that are not involved in catalytic process should be engaged in covalent bonding procedures.

Figure 10: Immobilization of antibodies on gold surface through covalent modifications

Another simple method of covalent immobilization in biosensor is cross-linking of enzymes or proteins. This is done through cross-linking agents such as glutaraldehyde (figure 11).

This technique involves the formation of covalent bonds between two proteins by using bifunctional glutaraldehyde containing reactive end groups that react with functional groups of proteins.

2.3.2. Biosensor performance attributes: Characteristics

Biosensors are said to be successful only when they fulfils certain criteria which are universal for their performance evaluation. First of all it is the linear range of detection that is considered valuable for estimating biosensors performance. Generally it is desired that the biosensor should be able to sense a linear range of analyte concentration which should include the permissible limit of the analyte too. For example if a biosensor is constructed for detection of a heavy metal there should be a linear change in its signal (current, absorbance, fluorescence ,etc) with increase in heavy metal concentration of the water sample. This generally gives a linear line curve when plotted against analyte concentration as depicted in figure 12.

Such linear curves tell us about the uniform performance of the biosensor. The regression equation y = 2.6226x + 2.7312 is used to calculate concentration of the analyte in unknown sample. The lowest concentration of the analyte detectable by the developed biosensor is called its lower limit of detection (LOD). It is always desirable that the LOD of the analyte should be lower than its permissible limit. For example for lead (Pb), 10 ppb is the permissible limit in drinking water set by WHO, so any biosensor developed for its detection should be able to sense below 10 ppb. LOD is an important attribute of a biosensor and directly relates the biosensor performance with desired goals. This also defines the sensitivity of the biosensor. The lower is the detection limit more is the sensitivity of the system. Now days the sensitivity of biosensors could be enhanced with the incorporation of nanomaterials such as gold, silver, graphite oxide nanoparticles which impart new characteristics to the bioreceptor and enhance their affinity towards the analyte.

Another term associated with biosensor performance is the selectivity. A biosensor is always constructed against a selective target and it should be highly specific for the same, in the presence of other analytes and interfering agents. For example a biosensor developed for Pb could only be said selective if it shows a marked difference in its response against Pb in the presence of all other heavy metals and organic interfering agents (figure 13).

The selectivity is also related to another attribute called reliability. The biosensor is said to be reliable if it reproduces its results against a selective analyte in diverse sample matrixes. If a biosensor is built to detect an analyte in serum samples, it should be able to recognise its target in serum samples in the presence of all other constituents of the serum. The reproducible result outcome for the same analyte with varying concentrations, in a complex matrix defines the reliability of the biosensor.

Fast response time is another attribute that defines the performance of a biosensor. Biosensors furnish quick results in a time span of few seconds to few minutes. This is achieved due to the high specificity and selectivity of the bioreceptor against the analyte that delivers a real time signal of their interaction. The regeneration of bioreceptor after providing a signal is a desired attribute which is dependent on response time in a way. Smaller will be the response time, easy it would be to regenerate the bioreceptor in its original form. As an example an enzyme based biosensor developed for heavy metal detection could be regenerated by keeping the enzyme electrode in EDTA solution for some time. EDTA is a metal chelator which helps in removal of heavy metals from the enzyme and refreshes the enzyme for next analysis. Similarly new regeneration methods could be worked out for other kind of bioreceptors.

Finally the storage stability and life span of a biosensor decides whether it could be called a disposable kind of biosensor or meant for repeated use. One time use kind are called disposable biosensors which generally include non-regenerable bioreceptors. Either developed for repeated use or as disposable devices, biosensors need to have good storage stability, so that they could be used after few days of their fabrication or can be transported to a new place for on-site analysis. Generally storage is done at low temperatures, but a biosensor stable at room temperature would be the highly desirable.

2.3.3 Biosensor characterization techniques

In case of electrochemical biosensors it is necessary to confirm that the bioreceptor has been firmly immobilized on the electrode surface. This is done by directly looking at the electrode surface at very high resolution of nanometer scale through scanning electron microscope (SEM) or transmission electron microscope (TEM). Both the techniques help us to visualize the electrode surface and give indication of bioreceptor attachment at the surface when seen in comparison to bare electrode (figure 14). These techniques also help us to know the structural morphology and dimensions of the bioreceptor and nanomaterial complexes immobilized at the electrode surface.

Second technique used to characterize electrode surface is FTIR, in which we get the indication of functional groups present on the electrode surface. This indirectly tells about the presence of protein or other bioreceptor on electrode surface. Every functional group has a characteristic wavelength at which its vibration or stretching is observed in a FTIR record. The presence of vibration bands at wavelengths designated to amide, amine, carboxyl etc confirms the presence of protein at electrode surface (figure 15).

In addition to these techniques there are a number of other techniques which are used to characterize the modified surface of an electrode of sensing platform, but the mentioned techniques would be enough here for basic introduction

Concluding Remarks

Biosensors are portable analytical tools composed of a biocomponent and a transducer. They produce measurable signals in the form of change in current, light, mass etc in response to an analyte and consequently called amperometric, optical or piezoelectric biosensors respectively. Any compound of biological origin which may specifically recognize an analyte and produce respective signal may act as the biocomponent. The compatibility of the biocomponent with the transducer and the method used for immobilization of biocomponent on transducer surface defines the sensitivity of a biosensor. A successful biosensor is the one that produce sensitive and specific signal in response to a target in minimum time.