Biosensors Based On Nanomaterials Biology Essay


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The emergence of nanotechnology is opening new horizons for the development of biosensors. A biosensor is a device, which uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues or cells to detect chemical compounds by electrical, thermal or optical signals. Nanomaterials acquire a great impact on development of biosensors because of their unique size and shape dependent optical properties, high surface energy and surface-to-volume ratio and tunable surface properties. Many kinds of nano-biosensors have been developed and used in a wide array of biomedical and other applications. Although it is impossible to survey this entire fast moving field, this mini review focuses on recent advances in nanomaterials based biosensors and summarizes the main functions of nanomaterials in biosensors.

Keywords: Biosensors, nanomaterials, nanoparticles, proteins.

1. Introduction

Biosensors are tools for the analysis of bio-material samples to gain an understanding of their bio-composition, structure and function by converting a biological response into an electrical signal. A biosensor consists of three components: (i) a biomolecule, which is a highly specific recognition element (bioreceptor), (ii) a transducer, such as an electrode (Mitsubayashi et al., 2003) , or an optical fiber (Xing et al., 2000) that converts the molecular recognition event into a quantifiable signal and (iii) a detector, which detects signals from the transducer. These signals are processed and transferred to a display or data storage device. The characteristic trait of a biosensor is the direct spatial contact between the bioreceptor and transducing element (Thevenot et al., 1999)

Signal transduction has been carried out with electrochemical, quartz crystal microbalance (QCM), optical absorption, fluorescence, surface plasmon resonance (SPR), radioactive, magnetic and other transducers (Kerman et al., 2004; Liu et al. 2004; Baldini et al., 2003; Taylor et al., 2004; Tian et al., 2004). Biomolecules (ligand), such as enzymes, antibodies, oligonucleotides, microorganisms, peptides and cells (Santoni et al., 1997; Grant et al., 2003; Giakoumaki et al., 2003; Mariotti et al., 2002; Tombelli et al., 2002; Chee et al., 2001; Katayama et al., 2000; Matsubara et al., 2004) are immobilized on a solid substrate by numerous steps and used to detect the presence of an analyte, such as enzymatic substrates, antigens, oligonucleotides and so on.

A flow chart showing the conceptual working of a biosensor is shown in figure 1. The analytical devices composed of a bioreceptor can directly be interfaced to a signal transducer which together relates the concentration of an analyte (or group of related analytes) to a measurable response. The bio-reaction converts the substrate to product. The transducer measures the physical change that occurs with reaction at the bioreceptor and then converts it to an electrical signal. The electrical signal from the transducer is often low and superimposed upon a relatively high and noisy baseline. The signal processing normally involves subtracting a 'reference' baseline signal, derived from a similar transducer without any biocatalytic membrane, from the sample signal. The resultant signal difference is then amplified and electronically filtered out the unwanted signal noise. The relatively slow nature of the biosensor response considerably eases the problem of electrical noise filtration. The analog signal produced at this stage is usually converted to a digital signal and passed to a microprocessor stage where the data is processed, converted to concentration units and output to a display device or data storage device.

2. Functions of Nanomaterials

Nanomaterials in the range 1-100 nm exhibit unique physical, chemical and electronic properties such as size and shape dependent optical properties, high surface energy and surface-to-volume ratio and tunable surface properties. These novel properties make them suitable for designing novel sensing devices. A wide variety of nanomaterials including metal nanoparticles, oxide nanoparticles, quantum dots, nanowires, nanoshells, carbon nanotubes (CNTs), and even composite nanoparticles have found useful applications in many kinds of biosensors for the diagnosis and monitoring of diseases, drug discovery, proteomics, environmental detection of biological agents and so on. Many types of nanomaterials of different sizes and compositions can play various roles in different sensing systems (Katz et al., 2004; Wang, 2003). Figure 2 shows some examples of nanomaterials, which have found potential applications in different biosensors. Figure 3(a) shows a scanning electron micrograph (SEM) of enzyme coated carbon nanotube, a miniaturized enzymatic biosensor for medical, environmental and chemical analysis. Figure 3(b) illustrates nanowire sensors for detecting biomarkers of cancer.

The important functions offered by nanomaterials include the immobilization of biomolecules, the catalysis of electrochemical reactions, enhancement of electron transfer between electrode surfaces and proteins, labelling of biomolecules and acting as reactant.

2.1 Immobilization of Biomolecules

Nanoparticles can adsorb biomolecules strongly, and help the immobilization of biomolecules in biosensor construction due to the large surface area and high surface free energy. While adsorption of biomolecules directly on the surfaces of bulk materials may cause denaturation and loss of bioactivity, the adsorption of such biomolecules on the surfaces of nanoparticles can retain their bioactivity because of the biocompatibility of nanoparticles. It is interesting to note that most of the nanoparticles carry charges and hence they can electrostatically adsorb biomolecules with different charges. Some nanoparticles can immobilize biomolecules by other interactions. For instance, gold nanoparticles can immobilize proteins through the covalent bonds formed between gold atoms and amine groups and cysteine residues of proteins (Gole et al., 2001; Gole et al., 2002; Liu et al., 2003). Among the nanomaterials, gold nano particles are frequently used for the immobilization of proteins. Crumbliss et al. (1992) have reported immobilization of several kinds of enzymes with gold nanoparticles and fabrication of different enzyme electrodes, and these enzyme electrodes retained enzymatic activity. Chen et al. (1999) attached gold nanoparticles to gold electrodes modified with cysteamine monolayer, and then immobilized horseradish peroxidase on these nanoparticles. The influence of nanoparticle size on the performance of the prepared biosensors was also studied. They found that nanoparticles with smaller size were more suitable for enzyme immobilization. Similar studies have also been reported for the construction of biosensors based on the immobilization of different proteins with gold nanoparticles, such as horseradish peroxidase, microperoxidase-11, tyrosinase and haemoglobin (Smyth et al., 2005; Luo et al., 2005; Patolsky et al., 1999; Liu et al., 2004; Gu et al., 2001; Limiao et al., 2010).

He et al. (2004) have reported that SiO2 nanoparticles are excellent matrices for enzyme immobilization due to their good biocompatibility and easy preparation. Hu et al. (2004) immobilized several heme proteins with SiO2 nanoparticles through the layer-by-layer assembly, and investigated the driving forces for the assembly procedure. Many nanoparticles like Pt, Ag, TiO2, ZrO2, Fe3O4 and MnO2 can be used for the immobilization of biomolecules.

Extensive studies are reported for the electrochemical immunosensors based on the immobilization of antigen or antibody with nanoparticles. Zhuo et al. (2005) developed a reagentless amperometric immunosensor based on the immobilization of a-1-fetoprotein antibody with gold nanoparticles, and the immunosensor exhibited good long-term stability. They also reported a label-free immunosensor for Japanese B encephalitis vaccine (Yuan et al., 2005) through the immobilization of related antibody with gold nanoparticles. As antibodies and antigens are both proteins, their immobilization mechanism with nanoparticles is the same as the immobilization of enzymes. Besides the gold nanoparticles, other nanoparticles such as silver (Tang et al., 2005) and silica (Wang et al., 2004) have also been reported for the immobilization of antibodies and antigens.

It is interesting that DNA can also be immobilized with nanoparticles for the construction of electrochemical DNA sensors. In order to immobilize DNA on the surfaces of nanoparticles, the DNA strands are often modified with special functional groups that can interact strongly with certain nanoparticles. Fang et al. (2001) immobilized the oligonucleotide with a mercaptohexyl group at the 51-phosphate end on the 16 nm diameter gold nanoparticles. The immobilization of DNA with silica nanoparticles was also studied (Chen et al., 2004), and enhanced immobilization results were obtained. Chen's group described a sensitive electrochemical DNA biosensor based on multi-walled carbon nanotubes (MWCNTs) functionalized with a carboxylic acid group for covalent DNA immobilization and hybridization detection (Niu et al., 2008). Zhang et al. (2008) reported an electrochemical DNA biosensor based on MWCNTs/nanostructural ZnO/chitosan composites for DNA immobilization and hybridization detection.

2.2 Catalysis of Electrochemical Reactions

Nanomaterials, especially metal nanoparticles show outstanding catalytic properties. The combination of the catalytic properties of nanoparticles with the unique properties of biosensors can result in the construction of highly sensitive sensor systems. The introduction of nanoparticles into biosensors can decrease overpotentials of many analytically important electrochemical reactions. Zhu et al. (2002) have reported a sensitive NO microsensor, which was developed through the modification of a platinum microelectrode with gold nanoparticles. The gold nanoparticles catalyze the electrochemical oxidation of NO with an overpotential decrease of about 250 mV.

Platinum nanoparticles also exhibit good catalytic properties and have been used in electrochemical analysis. Niwa et al. (2003) investigated a highly sensitive H2O2 sensor based on the modification of a carbon film electrode with platinum nanoparticles. The modified electrode exhibited sensitive response to H2O2 due to the catalytic oxidation of H2O2 by platinum nanoparticles, and the H2O2 oxidation peak potential at this electrode was about 170 mV lower than that at platinum bulk electrode. As H2O2 is the product of many enzymatic reactions, the proposed electrode has the potential application as an electrochemical biosensor for many substances. They have further developed an electrochemical sensor for sugar determination by replacing platinum nanoparticles with Ni nanoparticles (You et al., 2003). It was reported that a graphite-like carbon film electrode containing 0.8% highly dispersed Ni nanoparticles had excellent electrocatalytic ability with regard to the electrooxidation of sugars, such as glucose, fructose, sucrose and lactose. Compared with the Ni-bulk electrode, the proposed electrode exhibited a high oxidation current for the detection of sugars at comparatively low applied potentials, and the detection limits obtained were at least one order of magnitude lower. Electrochemical sensors based on the catalytic properties of other metal nanoparticles have also been reported, such as the application of copper nanoparticles for amino acid detection (Zen et al., 2004).

Some of the nonmetal nanoparticles also have very good catalytic properties (Chen et al., 2003). A carbon paste electrode doped with copper oxide nanoparticles was developed for the detection of amikacin based on the catalytic properties of the copper oxide nanoparticles, and the oxidation current of amikacin at the prepared electrode was about 40 times higher than that of a bulk copper oxide modified carbon paste electrode.

2.3 Enhancement of Electron Transfer

Electrical contact between redox-enzymes and electrodes is significant in the construction of enzyme electrodes. However, enzymes have no direct electrical communication with electrodes because the active centres of enzymes are surrounded by considerably thick insulating protein shells and hence, the electron transfer between electrodes and the active centers are blocked. The conductivity properties of nanoparticles, especially metal nanoparticles, are suitable for enhancing the electron transfer between the active centers of enzymes and electrodes acting as electron transfer 'mediators' or 'electrical wires'. Some nonmetal nanoparticles, such as oxide nanoparticles and semiconductor nanoparticles, can also enhance the electron transfer between proteins and electrodes (Martin-Palma et al., 2009). The effective enhancement of electron transfer depends on the conductivity of nanoparticles and arrangement between nanoparticles and biomolecules. It is interesting that creating defined and ordered arrangements of nanoparticles using nanotechnology is a promising approach to the construction of biosensors with greatly enhanced electron transfer properties.

Xiao et al. (2003) have reported the enhancement of electron transfer between enzyme and electrode with nanoparticles. The enzyme electrode exhibited very fast electron transfer between the enzyme redox centre and the electrode with the help of the gold nanoparticles, and the electron transfer rate constant was found to be about 5000 s-1, which is about seven times faster than that between glucose oxidase and its natural substrate, oxygen. Moreover, the enzyme electrode could be used for glucose detection without interference, as the effective electrical contacting made it insensitive to oxygen or common interferants, such as ascorbic acid. The electron transfer between other redox proteins and electrodes have also been revealed with the help of gold nanoparticles. These gold nanoparticles acted as a bridge to electron transfer between protein and electrode. Both silver and gold nanoparticles have good electrical conductivity and hence, can be used to enhance the electron transfer between proteins and electrodes. Liu et al. (2003a) combined silver nanoparticles with pyrolytic graphite electrodes, and then immobilized cytochrome c on these nanoparticles. The silver nanoparticles act as an electrical bridge that 'wires' the electron transfer between cytochrome c and the electrode, and the electron transfer rate was about 15.8 per second.

2.4 Labelling Biomolecules

The labelling of biomolecules, such as antigen, antibody and DNA with nanoparticles plays a vital role in developing sensitive electrochemical biosensors. Biomolecules labelled with nanoparticles can retain their bioactivity and interact with their counterparts, and based on the electrochemical detection of those nanoparticles the amount or concentration of analytes can be determined. Metal nanoparticle labels can be used in both immunosensors and DNA sensors (Dequaire et al., 2000; Authier et al., 2001; Cai et al., 2002; Cai et al., 2003). Gold nanoparticles are the most frequently used metal nanoparticles labels available. Semiconductor nanoparticles and oxide nanoparticles are also used as labels for biomolecules.

Dequaire et al. (2000) reported a sensitive electrochemical immunosensor for goat immunoglobulin G (IgG) based on gold nanoparticles label. The primary donkey anti-goat IgG was immobilized on a microwell surface and interacted with the goat IgG to be determined, and then gold nanoparticle- labeled donkey anti-goat IgG was added to conjugate with the analyte. The complex was treated with acidic bromine-bromide solution resulting in the oxidative dissolution of the gold nanoparticles. The solubilized gold ions were then electrochemically reduced and accumulated on the electrode and subsequently detected by anodic stripping voltammetry using carbon-based screen-printed electrodes. The combination of the sensitive detection of gold ions with anodic stripping voltammetry and the release of a large number of gold ions upon the dissolution of gold nanoparticles associated with a single recognition event provides an amplification path that allowed the detection of the goat IgG at a concentration of 3 pM. Employing a similar procedure, Authier et al. (2001) developed a sensitive DNA sensor based on the labelling of oligonucleotide with 20 nm gold nanoparticles, and the sensor could detect the 406-base human cytomegalovirus DNA sequence at a concentration of 5 pM.

An electrochemical DNA biosensor based on silver nanoparticles label could detect the target oligonucleotides at levels as low as 0.5 pM. Cai et al. (2003) labeled 5'-alkanethiol capped oligonucleotide probes with gold-coated copper core-shell nanoparticles, and developed an electrochemical DNA sensor based on the indirect determination of solubilized Cu2+ ions by anodic stripping voltammetry. Similarly, Wang et al. (2003) described a procedure for monitoring DNA hybridization based on electrochemical stripping detection of an iron tracer. They labeled the DNA probe with goldcoated iron core-shell nanoparticles and dissolved the iron containing nanoparticles following DNA hybridization, and the released iron ions were determined by cathodic stripping voltammetry in the presence of 1-nitroso-2- naphthol ligand and a bromate catalyst.

Semiconductor nanoparticles have been extensively used as labels in electrochemical biosensors, especially DNA sensors (Merkoci et al., 2005). Thiolated oligonucleotides labeled with CdS semiconductor nanoparticles were employed as tags for the detection of DNA hybridization events (Wang et al., 2003c). Dissolution of the CdS nanoparticles with 1M nitric acid, and the chronopotentiometric stripping measurements of the dissolved Cd2+ ions with a mercury film electrode provided the electrical signal for the DNA analysis. Based on a similar principle, Wang et al. (2003b) developed a method for the simultaneous analysis of different DNA targets. Three different nucleic acids were immobilized on three different kinds of magnetic particles and hybridized with different DNA targets, and then DNA probes labeled with different semiconductor nanoparticles, such as ZnS, CdS and PbS nanoparticles, were added and hybridized with their complementary DNA targets.

2.5 Acting as Reactant

Nanoparticles are chemically more active than their bulk counter part due to high surface energy. Making use of this property, novel electrochemical sensing systems can be constructed. Chen et al. (2004, 2005) have developed several electroanalytical systems based on the reactivity of MnO2. Glucose oxidase and MnO2 nanoparticles were coimmobilized on the gate of an ion-sensitive field effect transistor (ISFET), and the resulting glucose biosensor showed a significant pH increase at the sensitive membrane with increasing glucose concentration, which is essentially different from the pH changes of conventional ISFET-based glucose biosensors (Chen et al., 2004). The driving force for pH change in the proposed biosensor is due to the special reaction of MnO2 nanoparticles with H2O2 (Reaction 1).

MnO2 +H2O2 +2H+ Mn2+ + 2H2O + O2 (1)

The total reaction in the proposed glucose biosensor is given in Reaction 2:

-D-glucose + O2 + MnO2 + H+ Mn2 + D-gluconate + H2O (2)

Obviously, one hydrogen ion is consumed and no oxygen is needed in Reaction 2, which results in the novel response mechanism and extended dynamic range of the MnO2 nanoparticle-based glucose biosensor.

Based on a similar response mechanism, a sensitive biosensor for lactate was developed based on layer-by- layer assembly of MnO2 nanoparticles and lactate oxidase on an ISFET (Chen et al., 2005). Its response to lactate was about 50 times higher than that of the biosensor without MnO2 nanoparticles. Manganese dioxide nanoparticles can also react with ascorbic acid, and a sensitive ISFET-based ascorbic acid sensor can be constructed based on the reaction (Chen et al., 2004). Manganese dioxide nanoparticles are simply deposited on the gate of an ISFET, and its reaction with ascorbic acid may result production of hydroxyl ions. This ascorbic acid sensor is more stable and sensitive than the enzyme-based ISFET sensor, and it can be easily prepared and renewed. Moreover, the reaction of MnO2 nanoparticles with ascorbic acid is also used to eliminate interference in a glucose biosensor (Chen et al., 2004). It is interesting that properties of PbO2 and CeO2 are similar to that of MnO2, and nanoparticles based on PbO2 and CeO2 may also have special reactive properties which can be used to construct biosensors.

3. Conclusions

Nanomaterials based biosensors have already been successfully employed for highly sensitive and selective detection of biomolecules through various transducing approaches. Reviews of many kinds of nano-biosensors have been reported based on specific properties of different nanomaterials, such as semiconductor based biosensors (Xiao et al., 2003), carbon nanotube based biosensors (Wang, 2004), magnetic nanoparticles sensors (Koh and Josephson, 2009) optical biosensors (Haes et al., 2002), aptamer biosensors (Chiu and Huang, 2009; Beate et al., 2008) and label free biosensors (Vestergaard et al., 2007). Koh et al. (2009) reviewed the physics of magnetic nanoparticles and their use as relaxation switch assay sensors, relaxation sensors, and magnetoresistive sensors. They have illustrated the possible approaches to sensing of biological targets and summarized the latest results using the different sensing mechanisms on a variety of agents (viruses, bacteria, cancer cells). Kim et al. (2009) show that localized surface plasmon resonance (LPSR) can be used to detect biological binding on gold nano-islands. In order to increase the sensitivity of the technique, they functionalize their ligands with gold nanoparticles, allowing their target receptors to be large proteins. Qi et al. (2009) review electrogenerated chemiluminescence (ECL) in biosensors. They reviewed the principle of ECL detection and discussed the types of nanoparticles for which it is relevant and the procedures to biofunctionalize them and immbolize them on electrodes. Liu et al. (2009) reviewed how many of these types of sensors can be applied to a single biological problem: DNA hybridization. Because DNA is self-complementary and its strands can be denatured under relatively mild conditions, probing for specific DNA sequences is one of the most successful forms of biosensing. Taking advantages of the novel physical and chemical properties, improved biosensors can be constructed. This review has addressed recent advances in nanomaterials based biosensors and summarized the main functions of nanomaterials in biosensors.

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