1-1 Biosensors
Since the glucose sensor was reported by Clark and Lyons in 1962 [Clark and Lyons, 1962], which generally recognized as the first biosensor, many types of biosensor and their associated techniques have been studied and developed [Bergveld, 1996; Nakamura and Karube, 2003]. According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), the biosensor is the device that uses specific biological components detect of an analytic with a physicochemical detector component. Biosensors are useful tools for investigation of bio-molecular interactions [Boozer et al., 2004; Campbell et al., 2004; Bollmann et al., 2005; Buchmueller et al., 2005]. The interaction between the analyte and the biological component is designed to produce a physicochemical signal that can be measured by the transducer and can be converted into a measurable effect such as an electrical signal [Vo-Dinh and Cullum, 2000]. Figure 1-1 illustrates the conceptual principle of the biosensing process.
1-1-1 Analytical devices
The biosensor analytical devices combine a biological material (e.g., tissue,
microorganisms, organelles, cell, cell receptors, enzymes, antibodies, nucleic acids, etc.) [Cosnier et al., 2004; Davidson et al., 2004; Davis et al., 2005], intimately associated with or integrated within a physicochemical transducer which could be optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical, and that is primarily responsible for the display of the results in a user-friendly way [Cavalcanti et al., 2008].
1-1-2 Transducer
The transducer and the detector element play an important role in the detection process.
Depending upon the variety form of signal resulting from the interaction between the analyte and the biological element, the transducer can be classified into electrochemical, optical, piezoelectric, thermal, etc. (Table 1-1). Furthermore, novel types of transducers are constantly being developed for use in biosensors. The signal can be transformed into another signal that can be more easily measured and quantified. For a given
analyte-recognition element reaction, several transduction schemes may be applicable.
However, constraints may be imposed by the intended use [Feriotto et al., 2004; Fojta et al., 2004; Fu et al., 2005]. A transducer should be not only highly specific for the analyte of interest, it should be able to respond in the appropriate concentration range and have a moderately rapid response time. The transducer also should be reliable, able to be
miniaturized, and suitably designed for practical application [Foulds and Lowe, 1985; Alocilja and Muhammad-Tahir, 2008].
1-1-3 Bio-recognition element
Biosensors consist of bio-recognition system, typically enzymes or binding proteins, such as antibodies, immobilized onto the surface of physico-chemical transducers.
Immuno-sensors are often used to describe biosensors which use antibodies as their
bio-recognition system [Graham et al., 2004; Gronewold et al., 2005; Halder et al., 2005].
In addition to enzymes and antibodies, the bio-recognition system can also include nucleic acid, bacteria, single cell organisms, and even whole tissues of higher organisms.
Specific interactions between the target analyte and the complementary bio-recognition layer produce a physico-chemical change which is detected and may be measured by the transducer
[Knight, 2004; Krieg et al., 2004; Ladd et al., 2004]. In principle, any bio-molecule or molecular assembly that has the capability of recognizing the analyte can be used as a bio-receptor.
1-1-4 Applications of biosensors
Biosensors are used in increasingly broader ranges of application. The following describes some of the current applications, clinical diagnosis and biomedicine, farm and veterinary analysis, process control (fermentation control and analysis), food and drink production analysis, microbiology (bacterial and viral analysis), pharmaceutical and drug analysis, industrial effluent control, pollution control and monitoring, mining and toxic gases, and military applications. The applications of biosensors are listed in Table 1-2.
Biosensors are beginning to move from the proof-of-concept stage to field testing and commercialization in the United States, Europe, and Japan. Biosensors have several characteristic properties. The following section describes properties of biosensors [Cho et al., 2004; Li et al., 2005].
1-1-5 Properties of biosensors
Specificity: Like other bio-analytical methods (such as immuno-assays and enzyme assays), biosensors use a biologically derived compound as the sensing element. The advantage of biological sensing elements is their remarkable ability to distinguish between the analyte of interest and similar substances. With biosensors, it is possible to measure specific analytes with great accuracy.
Speed: One characteristic of biosensors that distinguishes them from other bio-analytical methods is that the analyte tracers or catalytic products can be directly and instantaneously
measured. There is no need to wait for results from lengthy procedures carried out in centralized laboratories.
Simplicity: The uniqueness of a biosensor is that the receptor and transducer are integrated into one single sensor. This combination enables the measurement of target analytes without using reagents. For example, the glucose concentration in a blood sample can be measured directly by a biosensor (which is made specifically for glucose measurement) by simply dipping the sensor in the sample. This is in contrast to the conventional assay in which many steps are used and each step may require a reagent to treat the sample.
Continuous monitoring capability: Another advantage of biosensors is that the
bio-analytical assays can regenerate and reuse the immobilized biological recognition element.
For enzyme-based biosensors, an immobilized enzyme can be used for repeated assays. This feature allows these devices to be used for continuous or multiple assays. By contrast, immunoassays, including enzyme-linked immunosorbent assay (ELISA), are typically based on irreversible binding and are thus used only once and discarded.
1-2 Gold nanoparticles
Nanoparticles, especially gold nanoparticles (AuNPs), have received great interests due to their attractive electronic, unique optical, thermal and physical properties as well as catalytic properties and potential applications in the fields of bio-nanotechnology (such as drug and gene delivery, and bioimaging) and in the rapidly developing area of biosensors [Park et al., 2002; Guo and Wang, 2007; Wang et al., 2008]. Therefore, the synthesis and characterization of AuNPs have attracted considerable attention. Furthermore, they have been proposed as future building blocks in nanotechnology [Persoons and Verbiest, 2006;
Zhao et al., 2008]. Table 1-3 shows the application of AuNPs.
Since the first synthesis report of AuNPs appeared about 150 years ago, numerous preparative methods leading to monodisperse particles of adjustable size and shape have surfaced. The common method for synthesis of AuNPs is wet chemical synthesis. Another method for the experimental generation of AuNPs is by sonolysis. The details of synthesis methods, characteristic, and application of AuNPs are as below:
1-2-1 Wet chemical synthesis techniques for small spherical particles
The common method for synthesis of AuNPs is wet chemical synthesis. In a typical synthesis of AuNPs, gold salts such as AuCl3 are reduced by the addition of a reducing agent which leads to the nucleation of Au ions to nanoparticles. Furthermore, a stabilizing agent is also required for stabilize the AuNPs. The size and shape of nanoparticles greatly influences their properties [Rao and Cheethama, 2001]. For example, spherical AuNPs exhibit a single plasmon resonance in the visible region of the spectrum, while rodlike particles exhibit a longitudinal and transversal plasmon resonance [Hutter and Fendler, 2002]. The common wet chemical synthesis methods of AuNPs including the citrate reduction method and the Brust method.
The citrate reduction method: The citrate reduction method was proposed by Turkevich in 1951 and this is the most well-known and simplest method for synthesizing gold colloids.
This method is used to produce modestly monodisperse spherical AuNPs involving the reduction of HAuCl4 by sodium citrate in water. A typical standard citrate reduction procedure to fabricate AuNPs with an average diameter of 20 nm is as follows [Frens 1973;
Grabor et al., 1997; Glomm, 2005; Persoons and Verbiest, 2006]:
Firstly, a solution of 100 mL 1 mM hydrogen tetrachloroaurate (HAuCl4) in water was
boiled in reflux conditions under vigorous stirring and secondly 10 mL of 38.8 mM aqueous sodium citrate was quickly added to the HAuCl4 solution. This reaction resulted in color changes of the originally yellow solution to dark blue/grey. After 2 min, the color of solution became wine-red, indicating the end of the reaction. This mixture was further stirred and boiled for 15 min and subsequently cooled to room temperature while stirring continuously. The resulted colloidal AuNPs are approximately spherical and have an overall negative surface charge due to the citrate coverage. In the reaction, the citrate ions reduce the gold salt HAuCl4 according to
3 (H2CCOOH)2C(OH)COO− + 2AuCl−4 ↔ 3 (H2CCOOH)2C=O + 2Au + 8Cl− + 3CO2 + 3H+
The gold colloids are stabilized by negatively charged citrate ions and chloride ions that are still present in the solution. The citrate is not only as a reductant but also as a kinetic stabilizer. Irreversible aggregation or coagulation is easily induced by addition of
electrolytes (e.g. KI, NaCl, and KNO3) to the solution. The AuNPs size can be control by changing the concentration of the added sodium citrate [Frens, 1973]. To synthesis larger particles, less sodium citrate should be added. However, the results are less reproducible, the larger particles are less monodisperse and the color of the solution is violet, indicating the importance of the citrate ions stabilizing the gold colloids [Glomm, 2005; Persoons and Verbiest, 2006]. The AuNPs are stabilized by electrostatic repulsion due to adsorbed citrate ions on their surface that impart negative charge to the nanoparticles [Turkevich, 1985; Nath and Chilkoti, 2004].
The Brust method: This two-phase synthesis method was described by Brust and Schiffrin in 1994, and can be used to synthesize AuNPs in organic liquids that are normally not miscible with water [Brust et al., 1994].
In the Brust method, the gold colloids are sterically stabilized by organic molecules
having thiol, amide or acid groups in the solutions. The stabilization with organic molecules having thiols is due to the covalent bond that gold binds specifically to the sulfur atom of the thiol group [Rodriguez et al., 2003] while the organic molecules forms the actual stabilization preventing the particles to aggregate. The main advantage of the Brust method is that the AuNPs behaves like chemical compounds [Whyman, 1996]. The AuNPs can be precipitated, filtered off and redissolved in organic solutions. Furthermore, several stabilization agents with thiol, amide or acid groups can be used to sterically stabilize the gold colloids. The preparation processes is as follows [Brust et al., 1994]:
First, 30 mL of a 30 mM aqueous solution of HAuCl4 was mixed with a solution of tetraoctylammonium bromide (TOAB or TOABr) in 80 mL 50 mM toluene (C6H5CH3) and stirred vigorously. After the tetrachloroaurate was transferred into the organic layer, the l70 mg dodecanethiol was then added to the organic phase. Second, 25 mL of a freshly prepared 0.4 M aqueous solution of sodium borohydride (NaBH4) was slowly added with vigorous stirring. After further stirring for 3 hr the organic phase was separated, and evaporated to 10 mL in a rotary evaporator. To remove the excess of thiocholesterol, the organic phase was mixed with 400 mL ethanol. The mixture is then kept at -18°C for 4 hr and the dark brown precipitate was filtered off and washed with ethanol. The crude product was dissolved in 10 ml toluene and again precipitated with 400 ml ethanol. The overall reaction is as follows [Brust, 1994]:
AuCl4− (aq) + N(C8H17)4+ (toluene)
→
N(C8H17)4+ AuCl4− (toluene) mAuCl4− (toluene) + nC27H45SH (toluene) + 3me− →4mCl− (aq) + (Aum) (C27H45SH)n (toluene)
1-2-2 Synthesis of AuNPs by ultrasound
An alternative method for the fabrication of AuNPs is by ultrasound method. This
method can effectively form gold complexes and only after the addition of a suitable reducing agent to the sonicated solution will the formation of AuNPs be observed. The mechanism of the fabrication of the AuNPs depends on the pyrolysis of water and other organic compounds present in the aqueous solution resulting in the formation of free radicals at high temperatures and pressures. When water is sonicated in the presence of ethanol, the following reactions proceed [Okitsu et al., 2001; Caruso et al., 2002]:
H2O
→
•H + •OHCH3CH2OH + •H(•OH)
→
CH3CH•OH + H2 (H2O) CH3CH2OH→
pyrolysis radicalsThese radicals can reduce gold(III) ions into gold(II), gold(I), and finally gold(0).
When the AuCl4− is sonicated in water without the addition of ethanol, some AuNPs is
produced according to three separate near diffusion-controlled one-electron transfer steps with H• as the primary reducing species [Caruso et al, 2002]:
AuCl−4 + 3H•
→
Au(0) + 4Cl− + 3H+And in the presence of ethanol a more complex sequence of three separate one-electron transfer reactions may be summarized [Okitsu et al, 2001; Caruso et al, 2002]:
3CH3CH•OH + AuCl4−
→
3CH3CHO + Au(0) + 4Cl− + 3H+The reduction of AuCl4− to colloidal gold according to the above two reactions is simplified and the particle growth is much more complex in the real sample solution. The rate of gold(III) reduction can be controlled by the ultrasound irradiation conditions such as the temperature and the ultrasound intensity. The size of the AuNPs can be controlled by
changing the alcohol concentration and alkyl chain length [Caruso et al, 2002]. This method is useful in the rapid fabrication of AuNPs, but the particles are polydisperse which is a problem for applications where monodisperse solutions are required [Persoons and Verbiest, 2006].
1-2-3 Synthesis process of AuNPs
The synthesis process of AuNPs involves three distinct stages: nucleation, growth and coagulation [Turkevich, 1985; Goia and Matijevic, 1998; Goia and Matijevic, 1999].
In the first stage, nucleation, metal ions are reduced to metal atoms, and rapid collisions to form stable icosahedral nuclei of 1-2 nm in size. The factors that affect the initial
concentration of nuclei include the following: the concentration of the reducing agent, the solvent, temperature and reduction potential of the reaction. Increasing the molar ratio of reducing agent to metal salt causes rapid formation of a large number of nuclei and leads to smaller, monodisperse AuNPs. In contrast, decreasing the molar ratio leads to slow formation of a few nuclei, and results in larger AuNPs with a greater heterogeneity in size.
This stage is important for controling the shape, size and structure distribution of AuNPs and is typically complete in a few seconds. In the growth stage, the metal ions are reduced on the surface of the nuclei, until all the metal ions are consumed. The final stage for synthesis of AuNPs is coagulation, which involves prevention of AuNPs aggregation by the addition of stabilizing agents, which is either adsorbed or chemically bound to the surface of the AuNPs, and is typically charged. The equally charged AuNPs repel each other so that they are colloidally stable [Sperling et al., 2008].
1-3 Surface modification and bioconjugation of AuNPs
AuNPs are surrounded by a shell of stabilizing molecules. One end of these stabilizing molecules are either adsorbed or chemically linked to the gold surface, while the other end points towards the solution and provides colloidal stability [Sperling et al., 2008]. After synthesis of AuNPs, the stabilizer molecules can be replaced by other stabilizer molecules in a
ligand exchange reaction [Pellegrino et al., 2005].
Biological molecules can be attached to the AuNPs in several ways. If the biological molecules have a functional group which can bind to the gold surface (e.g., -SH, -CN, -NH2, -COOH, -OH), the biological molecules can replace some of the original stabilizer molecules when they are added directly to the AuNPs solution [Grabar et al., 1995; Kumar et al., 2004;
Sperling et al., 2008]. By choosing the suitable molecules, it is possible to adjust the surface properties of the particles and attach different kinds of molecules to the AuNPs.
1-4 Surface plasmon resonance of AuNPs
AuNPs have emerged as important colorimetric reporters because of their high
extinction coefficients and strongly distance-dependent optical properties [Kim et al., 2001;
Huang et al., 2005], the interesting optical and electronic properties that have served as a versatile platform for exploring many facets of basic science. AuNPs can strongly absorb and scatter visible light. When visible light shines on AuNPs, the light of a resonant
wavelength is absorbed by AuNPs and the visible light energy excites the free electrons in the AuNPs. The phenomenon induces surface electron oscillation of AuNPs and is responsible for the intense colors exhibited of AuNPs, the so-called surface plasmon resonance (SPR) [Sönnichsen et al., 2002; Nath and Chilkoti, 2004; Sperling et al., 2008; Zhao et al., 2008].
The schematic presentation of the SPR is shown in Figure 1-2. And the SPR depends strongly on the size, shape, medium, and the relative distance of the AuNPs [Su et al., 2003;
Sun and Xia, 2003; Schultz, 2003]. The Figure 1-3 shows UV-Vis absorption spectra of AuNPs with different diameters.
Small AuNPs (e.g., 13 nm in diameter) absorb green light, which corresponds to a strong
absorption band (surface plasmon band) at about 520 nm in the visible light spectrum, resulting in the AuNPs display red in color. When the AuNPs are small, the surface electrons are oscillated by the incoming light in a dipole mode. As the size of AuNPs increases, the light can no longer polarize the nanoparticles homogeneously. Hence, the higher order modes at lower energy dominate. This causes a red-shift and broadening of the surface plasmon resonances. Therefore, the surface plasmon band red shifts with increasing AuNPs size [Ghosh and Pal, 2007; Zhao et al., 2008]. The red shift and color change also can be observed during the aggregation of small AuNPs. The spherical AuNPs with
interparticle distance higher than the average particle diameter appear red in color. When the interparticle distance become smaller than the average particle diameter, their surface
plasmons combine (interparticle plasmon coupling), and the aggregate could be considered as a single large particle, resulting in color changes from red to blue [Link and El-Sayed, 1999;
Jena and Raj, 2008; Zhao et al., 2008]. The interparticle plasmon coupling can generate a huge absorption band shift (up to ~300 nm), and the color change can be observed by the naked eye. Therefore, complicated instruments are not required for analysis.
This unique optical property of AuNps can provide an elegant colorimetric platform for detection biological molecular interaction. When target analyte or a biological molecular which directly or indirectly triggers AuNPs aggregation (or redispersion of an aggregate), this process can be detected by the color change of the AuNP solution. The ratio of the
absorbances at 520 nm (for 13 nm AuNPs), which corresponds to dispersed particles, and a longer wavelength (e.g., 600 nm), which corresponds to aggregated particles, is often used to quantify the aggregation process or color change [Zhao et al., 2008].
1-5 Nanoparticles stability - DLVO theory
DLVO theory was developed by Derjaguin, Landau, Verwey and Overbeek in the 1940s, and has been used to explain the stability of colloids in suspension. The theory describes the force between charged surfaces interacting through a liquid medium. The stability of
colloidal system is determined by the balance between two opposing forces: electrostatic repulsion and van der Waals attraction [Craig et al., 1998; Malvern Instruments]. The particle interaction forces are described by Figure 1-4.
The dominant cause of aggregation is the van der Waals attractive forces between the particles, which are long-range forces [Shaw, 1980]. Van der Waals attraction is actually the result of attractive forces acting between individual particles in colloid system. The effect is accumulative. One particle of the first colloid has a van der Waals attraction to each particle in the second colloid. This phenomenon is repeated for each particle in the first colloid, and the sum of all of these is the total attractive forces. The variation in van der Waals force with distance between the particles is demonstrated by an attractive energy curve [Zeta-Meter, Inc.
1993; Pashley and Karaman, 2005].
The DLVO theory proposes that an energy barrier resulting from the repulsive force prevents particles aggregation. The particles will suspension in the solution, and the system will be stable. However, if the particles collide with sufficient energy to overcome that barrier, the attractive force will pull them approaching and adhering each other, the particles will be irreversibly aggregation. An electrostatic repulsion curve is used to indicate the energy that must be overcome if the particles are to be forced together. The maximum energy is related to the surface potential and the zeta potential. Therefore, the repulsive forces plays an important role in maintain the stability of the colloidal system. There are two essential mechanisms that affect dispersion stability.
Steric repulsion: The stability of colloidal dispersions can be enhanced by the addition of suitable material (protective agents) adsorbing or otherwise attaching to the particle surfaces and preventing the particle surfaces coming into close contact [Shaw, 1980; Persoons and Verbiest, 2006]. If enough material adsorbs to the particles, the coating is sufficient to keep
Steric repulsion: The stability of colloidal dispersions can be enhanced by the addition of suitable material (protective agents) adsorbing or otherwise attaching to the particle surfaces and preventing the particle surfaces coming into close contact [Shaw, 1980; Persoons and Verbiest, 2006]. If enough material adsorbs to the particles, the coating is sufficient to keep