**6. Quantitative detection of protein binding to the solid surface**

Proteins adsorb in differing quantities, densities, conformations, and orientations, depend‐ ing on the chemical and physical characteristics of the surface [87]. Protein adsorption is a complex process involving van der Waals, hydrophobic and electrostatic interactions, and hydrogen bonding. Although surface-protein interactions are not well understood, surface chemistry has been shown to play a fundamental role in protein adsorption. Moreover, the properties of protein over-layers can be altered by the underlying chemistry, which directly impinges on control of conformation and/or orientation [88].

During the past decade substantial progress has been made in understanding the mecha‐ nism of protein adsorption. Authors have reported a numerous of techniques, e.g. QCM [89, 90], surface plasmon resonance (SPR) [91,92], ellipsometry [91], FTIR [93], atomic force mi‐ croscopy (AFM) [94].

#### **6.1. Quartz crystal microbalance measurements**

certain molecular size and complexity are necessary: proteins with molecular weights great‐

The way in which an antigen and its antigen-specific antibody interact is analogues of a lock and key fit [66], by which specific geometrical configurations of a unique key enables it to

An antigen-specific antibody fits its special antigen in a highly specific manner, according to that the three-dimensional structures of antigen and antibody molecules are matching [70]. Antibody biosensors are interested wide and interesting group of sensing devices, which in‐ cludes i.e. Surface plasmon resonance [74], fiber-optic biosensor [75], magnetoelastic reso‐

Aptamers are folded single stranded DNA or RNA oligonucleotide sequences with the ca‐ pacity to recognize various target molecules. They are generated in the systematic evolution of ligands by exponential enrichment process which was first time reported by Ellington [78] and Tuerk [79]. In this way suitable binding sequences are first isolated from large oli‐ gonucleotide libraries and subsequently amplified. The main application for aptamers is in biosensors. While antibodies are used in ELISA, the similar process for aptamers is called ELONA (enzyme linked oligonucleotide assay). They have many advantages over antibod‐ ies such as easier deposition on sensing surfaces, higher reproducibility, longer shelf life, easier regeneration and a higher resistance to denaturation. As antibodies, they are charac‐

A receptor which is designed and fabricated and to mimic a bioreceptor is often defined as biomimetic receptor. According to the phenomena several different techniques have been developed over the years for the construction of biomimetic receptors [81,82]. These proce‐ dures include: genetically engineered molecules, artificial membrane fabrication and molec‐ ular imprinting method. The molecular imprinting method has existed as an attractive and

Artificial membrane fabrication for bioreception has been performed for many different ap‐ plications. Stevens *et al.* has developed an artificial membrane by incorporating gangliosides into a matrix of diacetylenic lipids [83]. The lipids were allowed to self-assemble into Lang‐ muir-Blodgett layers and were then photopolymerized *via* ultraviolet irradiation into poly‐ diacetylene membranes. However, molecular imprinting has been used for the construction

Cellular structures and cells have been used in the development of biosensors and bio‐ chips. These bioreceptors are either based on biorecognition by an entire cell/microorgan‐

terized by both, their high affinity and specificity to their targets [80].

accepted tool in developing an artificial recognition agents.

of a biosensor based on electrochemical detection of morphine [84].

er than 5000 Da are generally immunogenic.

50 State of the Art in Biosensors - General Aspects

nance sensor [76] and immunosensor [77].

open a lock.

**5.4. Aptamers**

**5.5. Biomimetic receptors**

**5.6. Cellular bioreceptors**

QCM (Quartz Crystal Microbalance, Fig. 7) technology enables studies of molecular interac‐ tions by measuring the weight of the molecules, much like a very sensitive scale or balance. When molecules are added to or removed from the sensor surface, it is detected as a change in the oscillation frequency of the sensor crystal; the change in resonance frequency is corre‐ lated to the change in mass on the surface. QCM technology does not have the same limita‐ tions with regard to surface proximity as other biosensor technologies, making it possible for the instrument to measure binding to large structures such as cells.

**Figure 7.** Quartz Crystal Microbalance - equivalent mechanical model; mass (M),a compliance (Cm), and a resistance. (rf). The compliance represents energy stored during oscillation and the resistance represents energy dissipation dur‐ ing oscillation

The method is very useful for monitoring the rate of deposition in thin film deposition sys‐ tems under vacuum. In liquid, it is highly effective at determining the affinity of molecules (i.e. proteins) to surfaces functionalized with recognition sites. Larger entities such as virus‐ es or polymers are investigated, as well. QCM has also been used to investigate interactions between biomolecules.

Upon protein adsorption to the crystal surface, the oscillatory motion of the crystal was dampened, causing a decrease in the resonant frequency. The frequency shift of the QCM is due to a change in total coupled mass, including water interaction within the protein layer. The *Sauerbrey* equation relates the measured frequency shift (Δ*f*) and the adsorbed mass (m) [95].

$$
\Delta f = -\frac{2\Delta m f\_0}{A\sqrt{\rho\_q \mu\_q}} = -\frac{2f\_0^{'2}}{A\sqrt{\rho\_q \mu\_q}}\Delta m \tag{1}
$$

where


The sensor can be used for the direct, marker-free measurement of specific interactions be‐ tween immobilized molecules and analytes in solution. Binding of a soluble analyte to the immobilized ligand causes a shift in the resonance frequency, and this signal can be record‐ ed using a frequency counter with high resolution. This method, despite its existence for four decades, has only recently been developed for immunological measurements in a flow through system [96].

In contrast to the optical techniques, which are not sensitive to water associated with adsor‐ bed proteins, the *f*-shift of the QCM is due to the change in total coupled mass, including hydrodynamically coupled water, water associated with the hydration layer of e.g. proteins and/or water trapped in cavities in the film [89].

A recent extension of the technique, called QCM-D, to simultaneously measure changes in the frequency, *Δf,* and in the energy dissipation, *ΔD*, of the QCM provides new insight into e.g. protein adsorption processes [89] as well as other surface-related processes.

#### **6.2. Surface plasmon resonance**

**Figure 7.** Quartz Crystal Microbalance - equivalent mechanical model; mass (M),a compliance (Cm), and a resistance. (rf). The compliance represents energy stored during oscillation and the resistance represents energy dissipation dur‐

The method is very useful for monitoring the rate of deposition in thin film deposition sys‐ tems under vacuum. In liquid, it is highly effective at determining the affinity of molecules (i.e. proteins) to surfaces functionalized with recognition sites. Larger entities such as virus‐ es or polymers are investigated, as well. QCM has also been used to investigate interactions

Upon protein adsorption to the crystal surface, the oscillatory motion of the crystal was dampened, causing a decrease in the resonant frequency. The frequency shift of the QCM is due to a change in total coupled mass, including water interaction within the protein layer. The *Sauerbrey* equation relates the measured frequency shift (Δ*f*) and the adsorbed

> 2 2 0 0 2 2 *qq qq mf f f m A A* rm

D

)

)

 rm

D =- =- D (1)

)

ing oscillation

between biomolecules.

52 State of the Art in Biosensors - General Aspects

**•** *f0* – Resonant frequency (Hz) **•** *Δf* – Frequency change (Hz)

**•** *A* – Area between electrodes (cm2

**•** *ρq* – Density of quartz (ρq = 2.648 g/cm3

**•** *µq* – Shear modulus of quartz (µq = 2.947 x 1011 g/cm.S2

**•** *Δm* – Mass change (g)

mass (m) [95].

where

Surface plasmon resonance (Figure 8) can be applied as a convenient, sensitive and labelfree technique to study various surface phenomena. SPR is a surface sensitive, spectroscopic method which measures change in the thickness or refractive index of biomaterials at the in‐ terface between metal surfaces, usually a thin gold film (50–100 nm) coated on a glass slide, and an ambient medium. In SPR the test proteins are immobilized on a gold-surface, unla‐ belled query protein is added, and change in angle of reflection of light caused by binding of the probe to the immobilized protein is measured to characterize biomolecular interactions in the real-time [97].

**Figure 8.** Surface Plasmon Resonance

SPR has been used for many biomedical, food and environmental applications [98]. In exam‐ ple, Hiep *et al*. [99] developed a localized SPR immunosensor for detection of casein allergen in raw milk. There was also generated a unique SPR-based microarray using natural glycans for rapid screening of serum antibody profiles [100]. SPR microarrays was utilized in combi‐ nation with HT antibody purification technologies for rapid and proper affinity ranking of antibodies [101]. SPR-based biosensors are in great demand as they provide label-free, realtime detection of the biomolecular interactions.

#### **6.3. Ellipsometry**

Ellipsometry (ELM, Fig. 9) is an optical method that has been used extensively for protein adsorption studies. The method is based on the change upon protein adsorption of the state of polarization of elliptically polarized light reflected at a planar surface. From the changes in the ellipsometric angles (Δ, ψ), the refractive index and the thickness, morphology or roughness of the surface of layers can be deduced and used to determine e.g. the amount of adsorbed protein on a surface. Since the refractive index of adsorbed protein films is always close to *n*=1.5, which the film thickness can be calculated with quite good accuracy [102]. Moreover, the clear advantage of this technique is that the proteins under investigation re‐ quire no chemical treatments with markers etc. before use. Also, the measurement proce‐ dure is quite fast (on the order of a few seconds).

**Figure 9.** Ellipsometry setup

Since ellipsometry can be performed on most reflective substrates it can be easily conducted on an electrode surface and combined with electrochemistry. The ellipsometry and electro‐ chemical methods have been used to study protein adsorption on metal surfaces and specifi‐ cally human serum albumin on gold surfaces [103]. Chronoamperometry and ellipsometry were combined for the study of immunosensor interfaces based on methods of Immunoglo‐ bulin G adsorption onto mixed self-assembled monolayers [104]. The combined imaging el‐ lipsometry with electrochemistry have been employed to investigate the influence of electrostatic interaction on fibrinogen adsorption on gold surfaces [105].

#### **6.4. Atomic force microscopy**

SPR has been used for many biomedical, food and environmental applications [98]. In exam‐ ple, Hiep *et al*. [99] developed a localized SPR immunosensor for detection of casein allergen in raw milk. There was also generated a unique SPR-based microarray using natural glycans for rapid screening of serum antibody profiles [100]. SPR microarrays was utilized in combi‐ nation with HT antibody purification technologies for rapid and proper affinity ranking of antibodies [101]. SPR-based biosensors are in great demand as they provide label-free, real-

Ellipsometry (ELM, Fig. 9) is an optical method that has been used extensively for protein adsorption studies. The method is based on the change upon protein adsorption of the state of polarization of elliptically polarized light reflected at a planar surface. From the changes in the ellipsometric angles (Δ, ψ), the refractive index and the thickness, morphology or roughness of the surface of layers can be deduced and used to determine e.g. the amount of adsorbed protein on a surface. Since the refractive index of adsorbed protein films is always close to *n*=1.5, which the film thickness can be calculated with quite good accuracy [102]. Moreover, the clear advantage of this technique is that the proteins under investigation re‐ quire no chemical treatments with markers etc. before use. Also, the measurement proce‐

Sample

Since ellipsometry can be performed on most reflective substrates it can be easily conducted on an electrode surface and combined with electrochemistry. The ellipsometry and electro‐ chemical methods have been used to study protein adsorption on metal surfaces and specifi‐ cally human serum albumin on gold surfaces [103]. Chronoamperometry and ellipsometry were combined for the study of immunosensor interfaces based on methods of Immunoglo‐ bulin G adsorption onto mixed self-assembled monolayers [104]. The combined imaging el‐

Analyzer

Compensator

(optional) <sup>Φ</sup>

Detector

time detection of the biomolecular interactions.

54 State of the Art in Biosensors - General Aspects

dure is quite fast (on the order of a few seconds).

Polarizer

Compensator (optional)

Light source

**Figure 9.** Ellipsometry setup

**6.3. Ellipsometry**

Since its invention atomic force microscopy (AFM) has been a useful tool for imaging a wide class of biological specimens such as nucleic acids, proteins, and cells at nanometer resolu‐ tion in their native environment [106]. AFM can also be applied to measure intermolecular force based on the deflection signal of the AFM probe (cantilever) caused by the force be‐ tween the cantilever modified with a molecule of interest and a complementary molecule immobilized on a substrate.

Atomic force microscopy (AFM) is a very high-resolution type of scanning probe microsco‐ py, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit. Because the atomic force microscope relies on the forces between the tip and sample, knowing these forces is important for proper imag‐ ing. The force is not measured directly, but calculated by measuring the deflection of the lever, and knowing the stiffness of the cantilever. Hook's law gives:

$$F = -kz \tag{2}$$

where *F* is the force, *k* is the stiffness of the lever, and *z* is the distance the lever is bent.

The major advantage of AFM is to carry out measurements on non-conducting substrates, and to determine particle height as well, with atomic level precision and flexibility [107]. AFM is a useful device not only to trace topography of biological samples with molecular resolution under physiological conditions but also to study the interaction force between bio-molecular pairs and the mechanical properties of proteins at the single molecular level [108]. Moreover, the AFM is used as a manipulator to obtain DNA from chromosomes [109], or mRNA from local regions of living cells [110].

**Figure 10.** AFM image of immobilized invertase in LS lipid-like film; left – pure lipid film, right – hybrid protein film. All images are 3 µm x 3 µm [17]

Several measurements of the intermolecular force produced by biomolecular interaction were reported, i.e. the AFM surface topography of phospholipids LB films have shown a smooth surface. In presence of protein in LB film well-defined structures were observed, characterized by domains, globules, grains with different diameters. The images indicates that both enzyme molecules are not only properly entrapped in the composite membrane but also well exposed at the surface, which can be clearly seen in Figure 10. The recorded images show a relatively high homogeneity of the topography, especially in case of lipid film [17].

## **7. Prospects and future trends**

The advances observed in the areas of biochemistry, chemistry, electronics and bioelectron‐ ics will markedly influence future of biosensor production. Progresses in biosensors technol‐ ogy focus on two main aspects: transducer technology development and sensing element development [111]. New improved detection systems developed under the areas of micro‐ electronics or even nanoelectronics can be used in biosensors. However, since biosensor sen‐ sitivity and selectivity depend basically on the properties of the biorecognition elements, a crucial aspect in future biosensors is the development of improved molecular recognition el‐ ements. In this respect, biotechnology and genetic engineering offer the possibility of tailor binding molecules with predefined properties

According to fact, that miniaturization of devices as well as multi-sensor arrays are expected to have a marked impact in biosensors technology, the use of thin film methods for prepara‐ tion of the recognition layers provides a simple procedure for the functionalization of elec‐ trode surfaces using nanogram amounts of material. Different techniques can give either highly ordered or amorphous film, ensured a high level of control of the environment and often resemble the environment found inside biomembranes, thereby guaranteed the stabili‐ zation of biomolecules. Biosensors produced using these layer technologies can display high sensitivities, be easily interrogated using electronic, optical or mass-sensitive techniques, can often be regenerated and display good stability.

Another current trend is the combination of physics and biology in the creation of new nanostructures. Nanotechnology comprises a group of emerging techniques from physics, chemistry, biology, engineering and microelectronics that are capable of manipulating mat‐ ter at nanoscale. This novel technology bridges materials science, and biochemistry/chemis‐ try, where individual molecules are of major interest [112]. Inspired by nature, molecular self-assembly has been proposed for the synthesis of nanostructures capable to perform unique functions. According to that, novel tools that combine different sensing methods can provide also the necessary complementary information that is needed to understand the limitations and to optimize the performance of the new techniques. Therefore, introducing existing methods (e.g., SPR, QCM, ellipsometry) allow parallel complementary investiga‐ tions of the biochemical processes that take place at the interface between the devices and the biological sample.

At present, biosensor research is not only driving the ever-accelerating race to construct smaller, faster, cheaper and more efficient devices, but may also ultimately result in the suc‐ cessful integration of electronic and biological systems. Thus, the future development of highly sensitive, highly specific, multi-analysis, nanoscale biosensors and bioelectronics will require the combination of much interdisciplinary knowledge from areas such as: quantum, solid-state and surface physics, biology and bioengineering, surface biochemistry, medicine and electrical engineering. Any advancement in this field will have an effect on the future of diagnostics and health care.
