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Advances in biosensors: principle, architecture and applications 1

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Veeradasan Perumal, Uda Hashim 3

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Institute of Nano Electronic Engineering (INEE), University Malaysia Perlis (UniMAP), 5

Perlis, Malaysia 6

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Correspondence to: 9

Veeradasan Perumal, 10

Biomedical Nano Diagnostics Research Group, 11

Institute of Nano Electronic Engineering (INEE), 12

University Malaysia Perlis (UniMAP), 13

01000 Kangar Perlis, Malaysia 14

E-mail: veeradasan@hotmail.my 15

Tel: 04-9798580 16

Fax’s: 04-9798578 17

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Summary 21

The ability to detect pathogenic and physiologically relevant molecules in the body with high 22

sensitivity and specificity offers a powerful opportunity in early diagnosis and treatment of 23

diseases. Early detection and diagnosis can be used to greatly reduce the cost of patient care 24

associated with advanced stages of many diseases. However, despite their widespread clinical 25

use, these techniques have a number of potential limitations. For example, a number of 26

diagnostic devices have slow response times and are burdensome to patients. Furthermore, 27

these assays are expensive and cost the health care industry billions of dollars every year. 28

Therefore, there is a need to develop more efficient and reliable sensing and detection 29

technologies. A biosensor is commonly defined as an analytical device that uses a biological 30

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recognition system to target molecules or macromolecules. Biosensors can be coupled to a 1

physiochemical transducer that converts this recognition into a detectable output signal. 2

Typically biosensors are comprised of three components: (1) the detector, which identifies the 3

stimulus; (2) the transducer, which converts this stimulus to a useful output; and (3) the signal 4

processing system, which involves amplification and display of the output in an appropriate 5

format. The goal of this combination is to utilize the high sensitivity and selectivity of 6

biological sensing for analytical purposes in various fields of research and technology. Here 7

we review some of the main advances in this field over the past few years, explore the 8

application prospects, and discuss the issues, approaches, and challenges, with the aim of 9

stimulating a broader interest in developing biosensors and improving their applications in 10

medical diagnosis. 11

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Key words: transducer; bioreceptor; enzyme; antibody; DNA; pathology, diagnosis 13

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INTRODUCTION 16

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Over the past decade, many important technological advances have provided us with the tools 18

and materials needed to construct biosensor devices. Since the first invention of Clark Oxygen 19

Electrode sensor, there are many improvements in sensitivity, selectivity, and multiplexing 20

capacity of modern biosensor. Before the various types of biosensor technologies and 21

application are discussed, it is first important to understand and define ‘biosensor’. A 22

biosensor according to IUPAC recommendations 1999, a biosensor is an independently 23

integrated receptor transducer device, which is capable of providing selective quantitative or 24

semi-quantitative analytical information using a biological recognition element (Thevenot et 25

al. 1999). Essentially it is an analytical device, which incorporating a biological or biological 26

derived recognition element to detect specific bio-analyte integrated with a transducer to 27

convert a biological signal into an electrical signal (Lowe 2007). The purpose of a biosensor 28

is to provide rapid, real-time, accurate and reliable information about the analyte of 29

interrogation. Ideally, it is a device that is capable of responding continuously, reversibly, and 30

does not perturb the sample. Biosensors have been envisioned to play a significant analytical 31

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role in medicine, agriculture, food safety, homeland security, bioprocessing, environmental 1

and industrial monitoring (Luong et al. 2008). A biosensor consists of three main elements, a 2

bioreceptor, a transducer and a signal processing system (David et al. 2008). A biological 3

recognition element or bioreceptor is generally consists of an immobilized biocomponent that 4

is able to detect the specific target analyte (Kahn & Plaxco 2010). This biocomponents are 5

mainly composed of antibodies, nucleic acids, enzyme, cell and etc. Transducer in the other 6

hand is a converter. The reaction between the analyte and bioreceptor bring about a chemical 7

changes such as the production of a new chemical, release of heat, flow of electrons and 8

changes in pH or mass. The biochemical signal is converted into an electrical signal by the 9

transducer. Some of the commonly used transducers have been discussed in 3.0. Eventually, 10

signal processing where the electrical signal is amplified and sent to a microelectronics and 11

data processor. A measurable signal is produced, such as digital display, print-out or optical 12

changes. Figure 1 shows a schematic diagram of the typical components in a biosensor. There 13

is a need for simple, rapid and reagentless method for specific determination, both qualitative 14

and quantitative, of various compounds in various applications (Shantilatha et al. 2003). 15

Hence, it is paramount to have fast and accurate chemical intelligence which particularly 16

conspicuous in human health care. 17

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Fig. 1.0. Biosensor operation. 19

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CLASSIFICATION OF BIOSENSOR 1

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Biosensors can be classified either by the type of biological signaling mechanism they utilize 3

or by the type of signal transduction they employ. Figure 2.0 shows the different categories of 4

biosensor. 5

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Fig. 2.0. Different categories of biosensor. 9

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BASED ON BIOLOGICAL SIGNAL 12

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The bioreceptor or biological recognition element is the significant distinguishing feature of a 14

biosensor. The bioreceptor compromises the recognition system of a sensor towards the target 15

analyte. Essentially it is crucial for a bioreceptor to be selective and sensitive towards the 16

specific target analyte to prevent the interference by other substance from sample matrix 17

(Lowe 2007). Generally biosensors can be classified by the type of biological signaling 18

mechanism they utilize. The biological signaling used by biosensors can be divided into five 19

major mechanisms (Fig. 3.0). Here, we will discuss each of these mechanisms in detail and 20

their application: 21

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Fig. 3.0. Methods of biosensing with various biological signal mechanism: (a) antibody/antigen; (b) 2

enzyme catalyze; (c) nucleic acid; (d) cell-based; (e) biomimetic. 3

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Enzyme based sensor 5

Enzyme based biosensor are the earliest biosensor among all the other biosensor, these 6

biosensor is first introduced by Clark and Lyons in 1962 an amperometric enzyme electrode 7

for glucose sensor which use ‘soluble’ enzyme electrode (Shantilatha et al. 2003). Since the 8

first biosensor, enzyme based biosensor has face a massive growth in usage for various 9

application till present. Enzymes are very efficient biocatalysts, which have the ability to 10

specifically recognize their substrates and to catalyze their transformation. These unique 11

properties make the enzymes powerful tools to develop analytical devices (Leca-Bouvier and 12

Blum 2010). Enzyme-based biosensors associate intimately a biocatalyst-containing sensing 13

layer with a transducer. Enzyme based biosensor working principal is based on catalytic 14

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action and binding capabilities for specific detection (David et al. 2008). The enzyme based 1

biosensors were made of enzyme as bioreceptor which is specific to detect targeted analyte 2

form sample matrix. The lock and key and induced fit hypothesis can apply to explain the 3

mechanism of enzyme action which is highly specific for this type of biosensor. This specific 4

catalytic reaction of the enzyme provides these types of biosensor with the ability to detect 5

much lower limits than with normal binding techniques. This high specificity of enzyme–6

substrate interactions and the usually high turnover rates of biocatalysts are the origin of 7

sensitive and specific enzyme-based biosensor devices (Leca-Bouvier and Blum 2010). 8

Ideally enzyme catalytic action can be influence by several factors such as the concentration 9

of the substrate, temperature, presence of competitive and non-competitive inhibitor and pH 10

(Cass 2007). Essentially the Michaelis-Menten equation can be used to further explain the 11

detection limit of enzyme based biosensor (Parkinson and Pejcic 2005). Glucose oxidase 12

(GOD) and horseradish peroxidase (HRP) are the most widely used enzyme based biosensor 13

that has been reported in literature. However, some recent studies have shown that enzyme 14

based biosensor can be used to detect cholesterol, food safety and environmental monitoring, 15

heavy metals and also Pesticides (Amine et al 2006, Zapp et al. 2011, Nomngongo et al. 2012, 16

Soldatkin et al. 2012, Ju and Kandimalla 2008). Moreover, a recent studies reported the used 17

of enzyme catalytic implication incorporating with nucleic acid biosensor for DNA detection 18

(He et al. 2011, Lin et al. 2011). 19

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Immunosensors 21

An antibodies based biosensor was applied for the first time to detection in the 1950s, opening 22

the doors to the possibility of immuno-diagnosis (Donahue and Albitar 2010). Since then, 23

there have been vigorous effort made to develop immunosensor which composed of 24

antigen/antibody as bioreceptor as a tool for clinical diagnostics (Conroy et al 2009; Orazio 25

2011). An antibody is ‘Y’ shaped immunoglobin (Ig) that is made up of two heavy chains (H) 26

and two light chains (L). However some of human antibodies form dimeric or pentameric 27

structure by utilizing disulphide bonds and an extra protein called the joining or J- chain 28

(Wood 2006, Pohanka 2009). Each of the chain has a constant and variable part. The variable 29

part is specific to the antigen that is bind with corresponding antigen which is highly specific 30

and selective (Conroy et al 2009, Donahue and Albitar 2010). Hence, an immunosensor which 31

composed of antigen as bioreceptor utilizes the ability of antibody to bind with corresponding 32

antigen which is highly specific, stable, and versatile. The specificity of an antibody towards 33

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the binding side of its antigen is a function of its amio acids (Fowler et al. 2008). Those days, 1

there are two type of detection method which frequently used in immunosensor which are 2

optical and electrochemical. However optical detection transduction method has suffered from 3

poor sensitivity when coupled with radioimmunoassay, the short half-life of radioactive 4

agents, concerns of health hazards, and disposal problems. Electrochemical detection 5

overcomes problems associated with other modes of detection of immunoassays and 6

immunosensors. In contrast, electrochemical immunoassays and immunosensors enable fast, 7

simple, and economical detection that is free of these problems (Fowler et al. 2008). 8

However, recent advance in science and technology has created an optical transduction 9

method a new path towards highly sophisticated automated instrument. Hence, Optical and 10

electrochemical detection method are gaining mutual importance for development of 11

immunosensor (Shankaran et al. 2007, Bhatta et al. 2010). According to Ramirez et al. 2009, 12

Immunosensors have been envisioned to play an important role in the improvement of public 13

health by providing applications for which rapid detection, high sensitivity, and specificity are 14

important, in areas such as clinical chemistry, food quality, and environmental monitoring. 15

The development of immunosensor for bacteria and pathogen detection has gained a great 16

deal of attention due to its application in the point of care measurement (POC) (Skottrup et al. 17

2008, Barton et al. 2009, Braiek et al. 2012, Holford et al. 2012). Some recent studies shown 18

that immunosensor is widely explored toward the detection of cancer/tumor. Since the 19

traditional diagnostics method is poor in sensitivity, selectivity and time consuming, 20

immunosensor are become promising tools for cancer detection at early stages of cancer 21

[Ushaa et al. 2011]. 22

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DNA/Nucleic acid sensor 24

The use of nucleic acids sequence for the specific diagnostics application has developed since 25

the early 1953 and still growing widely (Liu A. et al. 2012). The highly specific affinity 26

binding’s reaction between two single strand DNA (ssDNA) chains to form double stranded 27

DNA (dsDNA) is utilized in nucleic acids based biosensor which appoint the nucleic acids as 28

biological recognition element. This method has promoted the development of DNA based 29

sensor from the traditional method such as coupling of electrophoretic separations and radio 30

iso-tropic which are high cost, hazardous, time consuming and etc. (Parkinson and Pejcic 31

2005). This biosensor working principal is based on recognition of the complementary strand 32

by ssDNA to form stable hydrogen bond between two nucleic acids to become dsDNA. In 33

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order to achieve this, an immobilized ssDNA is used as probe in bioreceptor which the base 1

sequence is complementary to the target of interest. Exposure of target to the probe which 2

results in hybridization of complementary ssDNA to form dsDNA will result in producing 3

biochemical reaction that allows transducer amplified the signal into electrical one. 4

Subsequently, literature shows that the present of some linker such as thiol or biotin is needed 5

in the effort to immobilize the ssDNA onto the sensing surface (Cagnin et al. 2009, Larzerges 6

et al. 2012). An important property of DNA is that the nucleic acid ligands can be denatured 7

to reverse binding and the regenerated by controlling buffer ion concentration (Parkinson and 8

Pejcic 2005). The nucleic acid biological recognition layer which incorporates with transducer 9

is easily synthesizable, highly specific and reusable after thermal melting o the DNA duplex 10

(Teles and Fonseca 2008). In addition, this biosensor possesses a remarkable specificity to 11

provide analytical tools that can measure the presence of a single molecule species in a 12

complex mixture (Brett 2005). DNA based biosensor has potential application in clinical 13

diagnostic for virus and disease detection (Chua et al. 2011, Lui C. 2009, Thuy et al. 2012). 14

Moreover, Yeh et al. 2012 recently has reported optical biochip for bacteria detection based 15

on DNA hybridization with detection limit of 8.25 ng/ml. However, electrochemical 16

transduction is most abandon method used to studying DNA damage and interaction which 17

reported in literature. The development of electrochemical DNA biosensor has received a 18

great deal of attention lately and this has largely been driven by the need to developed rapid 19

response, high sensitivity, good selectivity and experimental convenience (Liu A. et al. 2012). 20

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Cell based sensor 22

Cell based sensor are the type of biosensor, which use living cell as the biospecific sensing 23

element and are based on the ability of living cell to detect the intracellular and extracellular 24

microenvironment condition, physiological parameter and produces response through the 25

interaction between stimulus and cell (Wang and Liu 2010). Microorganisms such as bacteria 26

and fungi can be used as biosensors to detect specific molecules or the overall ‘‘state’’ of the 27

surrounding environment (Acha et al. 2010). Furthermore, proteins that are present in cells 28

can also be used as bioreceptors for the detection of specific analyte (Acha et al. 2010). 29

Essentially, living cell based biosensor is a unique biosensor in contrast to other type of 30

biosensor that contains materials that extracted from living things. These types of biosensor 31

has leveraged of pros and cons. The detection limit of this biosensor is mainly determined by 32

the natural environmental conditions in which the cell can stay alive for long period where 33

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need the control the physical and chemical parameter of environment. However the major 1

limitation with cell based biosensor are the stability of the cell, which depends on various 2

conditions such as the sterilization, lifetime, biocompatibility and etc. Another issue that 3

governs the success of a cell based sensor are depends primarily ion selectivity, in which cell 4

based sensor has poor selectivity of microbial sensor due to the multireceptor behavior of the 5

intact cells (Belkin and Gu 2010). Despite these pitfalls the cell based biosensor still favorable 6

among the researcher due to the advantages over the enzymes based biosensor. The cell based 7

biosensor are less sensitive to inhibition by solutes and are more tolerant of suboptimal pH 8

and temperature values than enzyme based biosensor, though they must not exceed the narrow 9

range in case of the cells dying, a longer lifetime can be expected than with the enzymatic 10

sensors and they are much cheaper because active cells do not need to be isolated (Struss et al. 11

2010). Literature revealed that, cell based sensor have become an emerging tools for medical 12

diagnostics (i.e. such as disease detection), environmental analysis, food quality control, 13

chemical-pharmaceutical industry and drugs detection (Veiseh et al. 2007, Banerjee and 14

Bhunia 2009, Banerjee and Bhunia 2010, Wang, T et al. 2010, Melamed et al. 2012, Bohrn et 15

al. 2012, Liu, Q et al. 2012 ). Similarly, Shinde et al. 2012 conclude that cell based biosensor 16

is envisioned to be an emerging frontier in the area of nano-diagnotics due to their attractive 17

characteristic. 18

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Biomimetic sensor 20

A biomimetic biosensor is an artificial or synthetic sensor that mimics the function of a 21

natural biosensor. These can include aptasensors, where aptasensors use aptamers as the 22

biocomponent (David et al. 2008). Aptamers were reported for the first time in the early 23

1990s where described as artificial nucleic acid ligands. Aptamers were thus chemically 24

related to nucleic acid probes, but behaved more like antibody and showing surprising 25

versatility compared to other bio-recognition components (Schneider et al 2010, Brys et al. 26

2007). Aptamer are synthetic strands of nucleic acid that can be designed to recognize amino 27

acids, oligosaccharides, peptides, and proteins. An aptamer has few advantages over antibody 28

based biosensor such as high binding efficiency, avoiding the use of animal (i.e reduced 29

ethical problem), smaller and less complex, and etc. However, common challenge facing 30

aptasensor is that they inherent the properties of nucleic acids such as structural pleomorphic 31

and chemical simplicity which reduced the assay efficiency and also increase its production 32

cost. Subsequently, some effort has been directed towards characterization and optimization 33

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of aptamer to overcome this limitation. Aptamer properties such as their high specificity, 1

small size, modification and immobilization versatility, regenerability or conformational 2

change induced by the target binding have been successfully exploited to optimize a variety of 3

bio-sensing formats (Schneider et al 2010). Aptamer based biosensor has been widely used in 4

various application. Recently sufficient progress has been made in biomimetics sensor and 5

aptasensor for clinical application (Vallet-Regi and Arcos 2008). This including clinical 6

diagnostics to detect pathogen, virus and infectious disease (Strehlitz et al 2008, Toress-7

Chavolla and Alocilja 2009, Wang Y. Et al. 2012, Weng et al. 2012). 8

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BASED ON TRANSDUCTION 1

Biosensor are normally categorized according to the transduction method they employ (figure 2

2.0). The transducer is component of biosensor, which has important role in the signal 3

detection process. Hence transducer can be defined as a device that convert a wide range of 4

physical, chemical or biological effect into an electrical signal with high sensitivity and 5

minimum disturbance to the measurand (Lowe 2007). There are various number of transducer 6

methods has developed over the decade; however recent literature review has highlighted the 7

most common methods that available (figure 2.0). This group can be further divided into two 8

general categories; labeled and label free type, which label free type biosensor growing 9

further recently (Parkinson and Pejcic 2005). 10

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Fig. 4.0: Methods of biosensing with various transducer mechanisms: (a) Optical; (b) 1

Electrochemical; (c) Calorimetric - thermal; (d) Piezoelectric- mass sensitive. 2

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Electrochemical biosensor 4

Electrochemical sensor in which an electrode is used as the transduction element, represent an 5

important subclass of sensor. According to IUPAC recommendation 1999, an electrochemical 6

biosensor is self-contained integrated device, which is capable of providing specific 7

quantitative or semi-quantitative analytical information using a biological recognition element 8

(biochemical receptor) which is retained in direct spatial contact with an electrochemical 9

transduction element (Thevenot et al. 1999, 2001). Electrochemical biosensors measure the 10

current produced from oxidation and reduction reactions. This current produced can be 11

correlated to either the concentration of the electroactive species present or its rate of 12

production/consumption. The resulting electrical signal is related to the recognition process 13

by target and analyte, and is proportional to the analyte concentration. A review by Wang et 14

al. (2008) has revealed that electrochemical immunosensors are gradually increasing in 15

popularity in clinical analysis and this is partly due to improved sensor design. Similarly, 16

Maria et al. (2008) revealed that the electrochemical immunosensor is a promising alternative 17

compared to existing laboratory method. Wang (2006) has reported the use of 18

electrochemical-based device for point-of-care cancer diagnostics which has brought the 19

electrochemical biosensor to new level in clinical diagnostics. Subsequently, all this report 20

has reveal the electrochemical biosensor has advantages such as fast, simple, low cost, high 21

sensitivity, and relatively simple instrumentation (Grieshaber et al. 2008, Mono et al. 2012). 22

Depending upon the nature of electrochemical changes detection during a biorecognition 23

event, electrochemical biosensors fall into one of four categories: amperometric, 24

potentiometric, impedance and conductometric. 25

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Amperometric 27

Amperometric sensor are based on the measurement of current as a function of time resulting 28

from the oxidation and reduction of an electroactive species in a biochemical reaction that 29

mainly depends on the concentration of an analyte with a fixed potential. There are three type 30

of electrode that usually employed in a amperometric sensor; The working electrode which 31

usually made of gold (Au), carbon (c), platinum (Pt), A reference electrode usually silver or 32

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silver chloride (Ag/AgCl) which has a fixed potential that controlled the potential of the 1

working electrode and the third electrode which called the counter or auxiliary where included 2

sometime to help measure the current flow (Wang J. 1996, Wang Y. et al. 2008). As the 3

certain molecule are oxidized or reduced (redox reactions) at inert metal electrodes, electrons 4

are transferred from the analyte to the working electrode or to the analyte from the electrode 5

(Banica 2012). The direction of flow of electrons depends upon the properties of the analyte 6

which can be controlled by the electric potential applied to the working electrode. If the 7

working electrode is driven to a positive potential an oxidation reaction occurs, and the 8

current flow depends on the concentration of the electroactive species (analyte) diffusing to 9

the surface of the working electrode. Similarly, if the working electrode is driven to a 10

negative potential then a reduction reaction occurs (Parkinson and Pejcic 2005). A third 11

electrode called the counter electrode is often used to help measure the current flow. There are 12

three generation of biosensor : first generation biosensor where the normal product of the 13

reaction diffuses to the transducer and causes the electrical response, second generation 14

biosensors which involve specific “mediators” between the reaction and the transducer in 15

order to generate improved response, and third generation biosensors where the reaction itself 16

causes the response and no product or mediator diffusion is directly involved (Saxena and 17

Malhotra 2003, Wang Y. et al. 2008). The amperometric transduction can be integrated with 18

Enzyme, Nucleic acids and Immunosensor biological recognition element for various 19

application such as glucose detection, disease, environmental monitoring and etc. (Ivnitski et 20

al. 1998, Ivnitski et al. 2003, Amine et al. 2006, Wang J. et al. 2006, Wang Y. et al. 2008, 21

Fang et al. 2011). However, there are limitation using this biosensor where, the presence of 22

electroactive interference in sample matrix can cause the transducer generate the false current 23

reading (Rogers and Mascini 1999). There been various method identified to overcome this 24

limitation such as diluting the sample, coating the electrode with various polymer, change the 25

medium of analyte, adding mediator and etc. (Jelen et al 2002, Belluzo et al. 2007, Shinde et 26

al. 2012). 27

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Potentiometric 29

Potentiometric biosensors rely on the use of ion-selective electrode and ion-sensitive field 30

effect transistor for obtaining the analytical information. In such sensor, the biological 31

recognition element converts the recognition process into a potential signal to provide an 32

analytical signal. Potentiometric transduction is first reported in the year 1969 where the 33

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enzyme based sensor is used for urea detection (Schelfer and Shcubert 1992, Saxena and 1

Malhotra 2003). Potentiometric sensor consists of two electrode where working (or Indicator) 2

electrode to develops a variable potential from recognition process and reference electrode 3

(usually Ag/AgCl) which require to provide a constant half-cell potential. The working 4

principle of the potentiometric transduction is depends on the potential difference between the 5

indicator electrode and reference electrode which accumulate during recognition process in an 6

electrochemical cell when zero or negligible amount of current flow through the electrode. 7

The electrical potential difference or electromotive force (EMF) between two electrodes is 8

measured using a high impedance voltmeter. The working electrode is made of permselective 9

ion-conductive membrane which sometimes called an ion-selective electrode (ISE). The 10

measurement of potential response of an potentiometric device is governed by the Nernst 11

equation in which the logarithm of the concentration of the substance being measured is 12

proportional to the potential difference (Grieshaber et al. 2008, Wang Y. et al. 2008). 13

According to Bakker and Pretsch 2010 recent progress enabled development and application 14

of potentiometric sensors with limits of detection (LODs) in the range 10-8 to 10-11 M. These 15

LODs relate to total sample concentrations and are defined according to a definition unique to 16

potentiometric sensors. The ISEs has the largest number of application in clinical chemistry 17

particularly the determination of the biologically relevant electrolytes in physiological fluids, 18

as billions of measurement are performed each year all over the world (Makarychev-19

Mikhailov et al. 2008). This comes as no surprise considering that potentiometric transduction 20

mechanism is very attractive for the operation of biosensor due to its selectivity, simplicity, 21

rapid, low cost and maintenance free measurement. However, the device is still less sensitive 22

and often slower than the amperometric counterpart (Makarychev-Mikhailov et al. 2008). The 23

field effect transistor have been used for the fabrication of integrated biosensor and there are 24

many device derived from this principle such as ion-selective field effect transistor (ISFET), 25

electrolyte –insulator semiconductor (EIS), light addressable potentiometric sensor (LAPS), 26

CHEMFET, BioFET and ENFET (Saxena and Malhotra 2003). Essentially, the LAPS device 27

is an example optical/electrochemical hybrid technique which consists of n-type silicon doped 28

with phosphorus and an insulating layer that takes advantage of the photovoltaic effect in 29

present of light source as a light emitting diode (LED), flashes rapidly (Pohanka and Skladal 30

2008). A review by Koncki 2007 has revealed the application of potentiometric based 31

biosensor in biomedical analysis. The author concludes that bioaffinity-based biosensor using 32

potentiometric transduction is not reliable. However, LAPS devices has proved to be highly 33

successful for immunoassay and overcome the previous less sensitivity problem encounter by 34

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potentiometric transduction (Wang et al. 2010, Jia et al. 2012). Recently, Jia et al. 2012 has 1

developed graphene oxide modified light addressable potentiometric sensor for ssDNA with 2

detection limit (LOC) of 1 pM to 10 nM. 3

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Conductometric 5

In this method the analytical information is obtained by measuring of electrolyte conductivity, 6

which varies with the changes in the ionic species concentration. In other word, 7

conductometric based transduction provides information about the ability of an electrolyte 8

solutions to conduct an electric current between electrodes. The conductometric device made 9

of two electrodes which separated by certain distance or medium such as nanowire, etc. Most 10

of the work that is reported in the literature on conductometric sensor associated with 11

enzymes where the ionic strength, and thus the conductivity, of a solution between two 12

electrodes changes as a result of an enzymatic reaction. The AC supply is used to apply across 13

the electrode for conductivity measurement. Thus, the ionic composition changes and 14

provides a conductance which measure using an Ohmmeter. However, some recent studies 15

has shown the conductometric transduction employed in micro/nano electronic device such as 16

FET to directly monitor the changes in conductance of an electrode as a result of the 17

hybridization of DNA, complementary antibody-antigen pair, etc.(Arya et al. 2007, Hnaiein et 18

al. 2008, Tang et al. 2011). The major advantages of conductometric device are non-19

requirement of reference electrode, inexpensive, possibility of miniaturization and direct 20

electrical response (Grleshaber et al. 2008, Lee et al. 2012). Unfortunately, the 21

conductometric transduction measurement is an additive property, hence less sensitive 22

compared to the other electrochemical methods and strongly dependence of the response upon 23

buffer capacity (Wang 1996). A review by Arora 2012 revealed that, conductometric based 24

transducer have envisioned for detection of food borne pathogens. However, recent literature 25

shown that conductometric based transduction has received a great deal of attention and 26

overcome the previous less sensitivity problem. Subsequently, some research has been 27

directed toward the improvement of performance and sensitivity (Kannan et al. 2012). 28

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Electrical impedance spectroscopy 30

Electrical impedance spectroscopy (EIS) based transduction method is not typically used 31

electrochemical detection; however this method has only recently become popular tools for 32

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bioreceptor transduction. These method are known to be similar to electrochemical detection 1

an conductivity detection that scan the detection volume with an electrical frequency sweep, 2

in the range of 10khz and 10mhz (McGuinness and Verdonk 2009). Essentially, impedance 3

spectroscopy has major advantages on lower concentration detection. A recent study shows 4

the EIS based transduction has been employed in tumor growth detection in 100-600 fg/ml 5

with the sensitivity/detection limit of 100 fg/ml (Onur and Kemal 2011). The EIS type 6

measurement is suitable for real time monitoring since it is able to provide a label free or 7

reagentless detection (Parkinson and Pejcic 2005, Pejcic et al. 2006). In EIS measurement, the 8

sample is placed on the sensing device such as nanogap, and a controlled alternating voltage 9

is applied to the electrode, and the current flows through the sample are monitored. The 10

electrical impedance resulting from the sample is calculated as the ratio of voltage over 11

current. The resulting electrical impedance measurement has both a magnitude and a phase, a 12

complex number. For any time-varying voltage applied, the resulting current can be in phase 13

with the applied voltage (resistive behavior), or out of phase with it (capacitive behavior). The 14

EIS consists of 3 electrode system, a potentiostats and a frequency response analyzer (FRA). 15

The three electrode which are working electrode that provides the measurement of current, 16

counter electrode that provides current to cell and reference electrode for voltage 17

measurement. The potentiostats function as a high input impedance provider and to maintain 18

the voltage across the electrode (Barsoukov and Macdonald 2005). Eventually, the FRA is 19

incorporated to supply the excitation waveform and provide a very convenient, high precision, 20

wide band method of measuring the impedance (Barsoukov and Macdonald 2005). A recent 21

review by Yang and Bashir 2008 discussed progress and application of impedance biosensor 22

for foodborne pathogenic bacteria detection. Impedance spectroscopy has been widely used 23

by many research groups to detect cancer/tumor cell, virus, bacteria and pathogen (Bayoudh 24

et al. 2008, Diouani et al. 2008, Kukol et al. 2008, Hong and Jang 2012, Thi et al 2012, Ohno 25

et al. 2012). This biosensor can become a powerful tool in the imminent future for clinical 26

diagnostics (Pejcic and Marco 2006). 27

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Optical based biosensor 29

Over the past decades, optical biosensor had made rapid advances and has been applied in a 30

number of important area including food safety, security, lifescience, environmental 31

monitoring and medical (Tsoka et al. 1998, Vedrine et al. 2003, Johansson et al 2008, Bhatta 32

et al. 2010a, Bhatta et al 2012, Md Muslim et al 2012). In medical, optical transducer have 33

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been established in both routine medical diagnostic and for medical research application 1

(Caygill et al. 2010). The word “optrode” is a combination of the words “optical” and 2

“electrode” is sometimes used to define optic based device (Biran et al. 2008). This method of 3

transduction has employed in many class of biosensor due to the many different types of 4

spectroscopy such as absorption, fluorescence, phosphorescence, Raman, SERS, refraction 5

and dispersion spectroscopy (Abdulhalim et al. 2007). These transduction methods are 6

capable of measure different properties of target/analyte. Optical based biosensor able to 7

provide a label free, real time and parallel detection (Francia et al. 2005, Fan et al. 2008, 8

Bhatta et al. 2010b). The surface plasmon resonance or fluorescence which integrated with 9

optical fiber is most popular method available for optical based biosensing (Caygill et al 10

2010). It appears that sensor based on optical fiber principal has gaining the research interest 11

and being employed in biosensor studies for. 12

13

Surface plasmon resonance 14

Surface plasmon resonance (SPR) biosensor use surface plasmon waves (electromagnetic 15

wave) to detect changes when the target analyte interact with biorecognition element on the 16

sensor. In principle, when the SPR biosensor is exposed to any changes, it will induce 17

changes in the refractive index which used to measure or observed the reaction. The SPR 18

transducer are incorporate with biomolecule /biorecognition element which recognize and 19

able to interact with specific analyte (Mol et al. 2010). Hence when target analyte interact 20

with the immobilized biomolecule on the sensor surface, it produces a change in the refractive 21

index at the sensor surface (Mol et al. 2010). This, changes produce a variation in the 22

propagation constant of the surface plasmon wave and this variation is measure to produce 23

reading. A spectrophotometer is used to measure the absorption spectrum of sample. There 24

been various biorecognition element have been incorporates with SPR biosensor such as 25

protein, antibody-antigen, nucleic acids and enzyme (Endo et al. 2005, Mannelli et al 2006, 26

Nguyen et al. 2007, Nakamura et al. 2008, Park et al. 2009). An important feature of SPR 27

biosensor is that it is able to provide label-free sensing without radioactive and fluorescence 28

which makes it highly attractive for real time monitoring (Fan et al. 2008, Endo et al. 2008). 29

In addition, the SPR based transduction can be used to and interaction without exhibit any 30

special properties of fluorescence or characteristic absorption and scattering bands (Homola et 31

al. 2008). However, some reports suggest that these method has suffer from specificity due to 32

non-specific interaction with biorecognition element which wrongly correlated by these 33

18

biosensor (Homola et al. 2008). The SPR based transductions are not suitable for studying 1

small analytes. Because the SPR measures the mass of material binding to the sensor surface, 2

very small analytes (Mr

<1000) give very small responses (Merve 2001) . The recent 3

improvements in signal to noise ratio have made it possible to measure binding of such small 4

analytes (Merve 2001). SPR biosensors can effectively detect binding by molecules as small 5

as about 2 kDa, but smaller molecules generate insufficient changes in bound mass and so 6

cannot be directly measured adequately ( Mitchell and Wu 2010). To date, SPR has been 7

widely used in fundamental biological studies, health science research, drug discover, clinical 8

diagnosis and environmental and agriculture monitoring (Shankaran et al 2007). Several 9

articles have appeared in the literature reviewing the application of SPR based biosensor in 10

pathogen and disease detection (Skottrup et al. 2008, Caygil et al 2010). Recently, Springer et 11

al. 2010 demonstrated that the SPR based biosensor is able to detect short sequence of nucleic 12

acids (20 bases) characteristic for Escherichia coli down to femtomole level in 4 minute. 13

14

Chemiluminescence 15

Chemiluminescence can be describe as method of energy produce from chemical reaction 16

which produce a emission of light or usually describe as Luminescence (Li D. 2008). 17

Essentially, when a chemical reaction occur, the atom or molecule relaxes from excited state 18

to its ground state which then a luminescence is produce as side product of the reaction. 19

Hence, chemiluminescence can be used to detect specific biochemical reactions which occur 20

and this property has contributed for chemiluminescence based biosensor development. In the 21

chemiluminescence biosensor, the reaction between analyte and the immobilized biomolecule 22

which has been marked with chemiluminescence species will end in generating light as result 23

of biochemical reaction. This emitted light can be detected using a Photo Multiplier Tube 24

(PMT). A review by Dodeigne et al. 2000 and Zhang et al. 2005 has shown that 25

chemiluminescence is an emerging tool for diagnostics with extremely high sensitivity along 26

with the simple instrumentation, fast dynamic response properties, and wide calibration range. 27

Not with standings, chemiluminescence based transduction has been widely applied and 28

embraced for immunosensing and nucleic acid hybridization (Atias et al. 2009, Ding et al 29

2008, Holford et al. 2012, Guo et al. 2013). Similarly, a number of papers have shown that 30

chemiluminescence applications in clinical, pharmaceutical, environmental and food analysis 31

(Kricka 2003, Gámiz-gracia et al. 2009, Lara et al. 2010, Liu M. et al. 2010). These methods 32

were also incorporated with immunosensor and optical fiber for detection of dengue virus in 33

19

human (Atias et al. 2009). More importantly, it was revealed that this type of transduction has 1

detection limit of 5.5 × 10−13 M (Ding et al. 2008). However, chemiluminescence transduction 2

has few drawbacks such as less quantitative accuracy due to short lifetime and not suitable for 3

real time monitoring (Parkinson and Pejcic 2005, Zhang et al. 2005, Mathews et al. 2009). 4

5

Fluorescence 6

The term Luminescence as describe above is the product of atoms or molecule which relaxes 7

from excited state to its ground state. The various types of luminescence differ from the 8

source of energy to obtain the excited state. This energy can be supplied by electromagnetic 9

radiation (photoluminescence also termed as fluorescence or phosphorescence), by heat 10

(pyroluminescence), by frictional forces (triboluminescence), by electron impact 11

(cathodoluminescence) or by crystallization (crystalloluminescence) (Dodeigne et al. 2000). 12

Hence, the fluorescence requires external light source (short-wavelength light) to initiate the 13

electronic transitions in an atoms or molecule which then produce luminescence (Longer 14

wavelength light). Eventually, fluorescence based biosensor has incorporate with 15

flourochrome molecules which used to produce light during the biorecognition event (Daly 16

and McGrath 2003). Since most of the biological sensing element and most analyte does not 17

possess intrinsic spectral properties, the biorecognition event is transduced to optical signal by 18

coupling fluorescence an optically responsive reagents to the sensing elements (Biran et al. 19

2008). For example, the nucleic acid or antibodies is used to tag with flourochrome and 20

convert the hybridization interaction between two complementary DNA stands into an optical 21

signal (Ramanathan et al. 2005, Berdat et al. 2006, Schultz et al. 2008). In fact, fluorescence 22

technology has been widely applied and embraced for immunochemical sensing in the 23

medical field (Hong and Kang 2006, Moghissi et al. 2008). Fluorescence based biosensing 24

were optimized widely in environmental monitoring as reported by Védrine et al. 2003. 25

Nonetheless a recent article has been appear in the literature on the use of flourescene 26

marking DNA biochip for analysis of DNA- carcinogen adducts (Grubor et al. 2004). The 27

major drawbacks of fluorescence technology are additional complexity of time-resolved 28

instrumentation, in either the time or frequency domains or both and not suitable for real time 29

monitoring (Thompson 2008, Abdulhalim et al. 2007). 30

31

20

Optical fiber 1

Optical fiber or which sometimes called optrodes, has received considerable interest for 2

developing as biosensor particularly in lower detection limit (LOC) sensing application. 3

Optrodes based optical fiber biosensors are composed of few major components: a light 4

source; biorecognition element which immobilized; an optical fiber which responsible to 5

transmit the light and act as the substrate; and a detector (e.g., spectrophotometer) where the 6

output light signals is measure. Hence, when the target analyte interacts with biorecognition 7

elements at the surface of the fiber, a biochemical reaction takes place resulting in the changes 8

of optical properties. These changes can be collated to the analyte concentration. The light 9

source is transmitted through the fiber optic, where the biorecognition event takes place. The 10

same or different fiber is used to guide the output light to the detector. The current trends of 11

bio-sensing using optical fiber are gaining researcher interest due to its major advantages such 12

as miniaturized sensor with higher performances, fiber optical sensors with small size, high 13

sensitivity, fast response, high selectivity, and low detection limits (Lee B. et al. 2009, 14

Zhang,L. et al. 2011). Optical fiber based biosensor also have several advantages compared to 15

electrochemical and other biosensing such as higher sensitivity, safer, free from 16

electromagnetic interference, no reference electrode needed and suitable for real time 17

monitoring (Biran et al. 2008). In addition this type of transduction is flexible hence suitable 18

for remote sensing, single molecule detection and reagentless (Biran et al. 2008). However, 19

there are several drawbacks such as poor stability of biorecognition elements, sensitive to 20

ambient light and etc. (Biran et al. 2008). Most of the work that is reported in the literature on 21

optical fiber biosensor applications has employed the optic fiber with other optical method 22

(Atias et al. 2009, Jang et al. 2009, Huang et al. 2009, Cecchini et al. 2012). Furthermore, 23

these methods have been used for various biomolecule detection such as antigen and nucleic 24

acid (Brogan et al. 2005, Leung et al. 2007, Amin et al. 2012). Thus, explained the versatility 25

of optical fiber biosensing. The optical fiber based biosensing has widely being used pathogen 26

and virus detection (Atias et al. 2009, Huang et al. 2009, Caygill et al 2010). Leung et al. 27

2008 has reported label free detection of DNA hybridization for pathogen detection using 28

fiber optic biosensor. 29

30

Piezoelectric based biosensor 31

Piezoelectricity can be explain as a linear interaction between mechanical and electrical 32

systems in non-centric crystal or similar structure which first discover by Curie brothers in 33

21

1880 (Katzir 2006, Tichy et al. 2010). Essentially, the piezoelectric based biosensor working 1

on the principal that an oscillating crystal resonates at a natural resonance frequency (Steinem 2

and Janshoff 2007, Tichy et al. 2010). The fundamental elements in a biosensor are transducer 3

and biorecognition element. Hence, in piezoelectric biosensor the transducer is made of 4

piezoelectric material (e.g., quartz) and the biosensing material that coated on the 5

piezoelectric material which vibrate at the natural frequency. The frequency is control by the 6

external electrical signal which produces a certain value of current, when the target analyte is 7

exposed to the sensing material the attachment/ reaction will cause the frequency shift which 8

will produce changes in current reading that can be collated to the mass of the analyte of 9

interest. There are two main types of piezoelectric sensors: bulk wave (BW) and surface 10

acoustic wave (SAW). However, literature shows piezoelectric sensors are not receive much 11

attention and inferior compared to electrochemical and optical based biosensing. 12

13

Bulk wave (BW) and Surface acoustic wave (SAW) 14

The bulk wave, quartz crystal microbalance and surface acoustic wave transducer is 15

fundamentally based on the piezoelectric effect. The unique properties of piezoelectric 16

material are utilized in this type of sensing. Quartz is the most commonly used piezoelectric 17

since it is cheap, can be processed to yield single crystal and can withstand chemical, thermal 18

and mechanical stress; however, there is report that lithium niobate and lithium tantalate can 19

aslo be used (Tichy et al. 2010). A recent review has shown that this method is very appealing 20

when integrate with Microelectromechanical systems (MEMS) for biosensing application 21

(Nicu et al. 2005). In addition the review states that this type of transduction is suitable for 22

sensitive, portable and real time biosensing (Nicu et al. 2005). Piezoelectric transducer has 23

been widely applied and embraced for immunosensing application (Vaughan et al. 2007, 24

Serra et al. 2008, Tothill 2009). Some report suggests that the piezoelectric transducer is 25

suitable for DNA and protein detection with detection limit of 1 ng/cm2 (Nirschl et al. 2009). 26

Several article have appear in the literature reporting the use of piezoelectric sensor in various 27

application such as cholera toxin diagnostic detection, hepatitis B, hepatitis C, food borne 28

pathogen detection and etc. (Skládal et al. 2004, Yao et al 2008, Serra et al. 2008, Chen et al. 29

2008, Chen et al. 2010). More importantly, it was revealed that piezoelectric is very sensitive 30

method, noting that a detection limit of 8.6 pg/l was obtained for hepatitis B virus DNA and 31

25ng/mL for cholera toxin detection (Yao et al. 2008, Chen et al. 2010). The advantages 32

using this type of transduction is the real time monitoring, label free detection and simplicity 33

22

of use (Dell’Atti et al. 2006, Chen et al. 2010). However, there are some drawbacks need to 1

overcome such as specificity, sensitivity as well as interference reduction (Glynn and 2

O’Connor 2010). In addition, this type of transducer method involves format and calibration 3

requirement (Leca-Bouvier and Blum 2010). A recent review by Kim et al. 2011 has review 4

the principle and application of nano diagnostic for nanobiosensor. The review also 5

concluded, that application range of the quartz crystal has been gradually expanded, new 6

measuring techniques that use the quartz crystal as a transducer for chemical sensors and 7

biosensors have been also developed (Kim et al 2011). 8

9

Calorimetric based biosensor 10

First enzyme based biosensors introduce by Clark and Lyons in 1962 has inspired and attracts 11

researcher interest in developing calorimetric based transduction. In fact, almost all chemical 12

and biological reaction involves exchange of heat (Xie et al. 1999). Thus, the general idea of, 13

generation and absorption of heat resulting in all biochemical reaction has contributed to the 14

birth of calorimetric based biosensing device. Initially, the calorimetric transduction has been 15

employed for enzyme based sensor, and has subsequently been applied in cell and 16

immunosensor (Xie et al. 1999, Ahmad et al. 2010). The principal of calorimetric measured 17

the changes in temperature in the reaction between biorecognition element and a suitable 18

analyte. This change in temperature can be correlated to the amount of reactants consumed or 19

products formed (Xie et al. 1999). A review by Yakovleva et al has discuss that among all 20

various concept of thermal detection, enzyme thermistor (ET) has been research widely for 21

various application. The major advantages of this type of thermal detection are the stability, 22

increase sensitivity and possibility of miniaturation (Ahmad et al. 2010). In addition 23

calorimetric based biosensor can be easily miniaturised and integrated with microfluidic for 24

increased sensitivity (Zhang and Tadigadapa 2004). In the calorimetric device, the heat 25

change is measured using either a thermistor (usually metal oxide) or thermopile (usually 26

ceramic semiconductor). A review by Cooper 2003 has shown that this method is very 27

appealing for label free screening of biomolecule interaction. Some recent studies have shown 28

that this technique capable of rapidly detecting the DNA hybridization (Watterson et al. 2002, 29

Buurma and Haq 2008, Paul et al. 2010, Xi et al. 2010). Recently, calorimetric method also 30

has been used in food industry and environmental monitoring (Maskow et al. 2012; Kirchner 31

et al 2012). 32

23

1

CONCLUSION AND OUTLOOK 2

3

The past decade has seen great advancements in the field of biosensor along many fronts. This 4

dynamic tool has been applied in many area of life science research, health care, 5

environmental, food and military application. Biosensor technology has received heightened 6

interest over the past decade, since it is a promising candidate for lower detection limit with 7

rapid analysis time at relatively low cost. However, the review shows that there is a lot of 8

studies have been undertaken using indirect measurement with simple clean buffer solution 9

instead direct measurement for in situ real-sample monitoring which is more vital. 10

Technological advances have provided us with the tools and materials needed to construct 11

biochip which integrated with microfluidic system, probe, sampler, detector, amplifier and 12

logic circuitry. This biochip is a promising candidate for label free, reagentless, real time 13

monitoring, miniaturization and low cost application. For medical application, this cost 14

advantage will allow the development of extremely low cost, disposable biochips that can be 15

used for in-home medical diagnostics of diseases without the need of sending samples to a 16

laboratory for analysis which time consuming. 17

18

19

ACKNOWLEDGEMENT 20

21

Author Veeradasan Perumal thankfully acknowledges collaborators at the Institute of Nano 22

Electronic Engineering (INEE) at University Malaysia Perlis (UniMAP). This work was 23

supported by INEE at (UniMAP), through the Nano Technology project. The views expressed 24

in this publication are those of the authors and do not necessarily reflect the official view of 25

the funding agencies on the subject. The appreciation also goes to all the team members in the 26

Institute of Nanoelectronic Engineering especially in the Biomedical Nano Diagnostics 27

Research Group. 28

29

24

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