**7. LW biosensors state-of-the-art**

**6. LW packaging and flow cells**

300 State of the Art in Biosensors - General Aspects

approaches [53,75,76].

a)

Outlet

Transparent PMMA Aluminum

ments. c) PDMS square seal with pick end to minimize the contact area.

Flow cells are usually constructed with an inlet at one side, an outlet at the other, and the channels facing the sensor (see Figure 9). In most cases, a rubber seal is used for sealing, and in LW flow-cells additional absorbers made with rubber materials at the ends of the IDTs are recommended to improve the signal response (see Figure 9b). Factors such as the flow cell design, flow patterns and flow-through system influence the binding efficiency and the

The permittivity and the dielectric losses of the liquid medium, necessary in LW biosensors applications, influence the propagation of Love modes, since this medium acts as an addi‐ tional layer. When the liquid medium is deposited over the device surface, the presence of this medium over the IDTs modifies the electrodes transfer function [73]. Permittivity and dielectric losses of the liquid lead to a corresponding change of the IDT input admittance, which influences the amplitude of the signal. A flow–through cell is crucial to eliminate this electric influence of the liquid. Such flow-cell isolates the IDTs from the liquid, confining the liquid in the region between the IDTs (sensing area). LW device packaging or flow-cells gen‐ erally use walls to accomplish this purpose. These walls, when pressed onto the device sur‐ face, disturb the acoustic wave, resulting in an increase in overall loss and distortion of the sensor response. Walls must be designed to minimize the contact area in the acoustic path in order to obtain the minimum acoustic attenuation (see Figure 9c). It is known that the acous‐ tic wave is significantly affected when increasing the walls width [27] and that materials used for these walls play an important role. Hence, great care must be taken to ensure that the designed LW flow cells do not greatly perturb the acoustic signal. Recently, some re‐ searchers have explored different possibilities to achieve the packaging of LW sensors for fluidic applications [27,74] and other authors have being exploring different LW flow-cell

PDMS absorbers

Outlet

Sensing area PDMS seal

Inlet

PDMS seal

c) Inlet

LW sensor

**Figure 9.** A developed LW flow cell for immunosensors application designed and fabricated by the authors of this chapter [53]. a) Overview of the flow cell b) Microscope view through the PMMA of the LW sensor and flow cell ele‐

b)

course of binding kinetics, resulting in possible variations of the true results [72].

The first approaches employing LW for biochemical sensing were reported in 1992 by Ko‐ vacs et al. [77] and by Gizeli et al. [78], who first demonstrated the use of such devices as mass sensing biosensors in liquids. However, it was not until 1997 that LW acoustic devices were used to detect real-time antigen-antibody interactions in liquid media [46].

In 1999, a contactless LW device was built in order to protect electrodes from the conductive and chemically aggressive liquids used in biosensing [79]. The advantage of this technique is that no bonding wires are required.

In 2000, a dual channel LW device was used as a biosensor to simultaneously detect *Legion‐ ella* and *E. coli* by Howe and Harding [80]. In this approach a novel protocol for coating bac‐ teria on the sensor surface prior to addition of the antibody was introduced. Quantitative results were obtained for both species down to 106 cells/mL, within 3 h.

In 2003, a LW immunosensor was designed as a model for virus or bacteria detection in liq‐ uids (drinking or bathing water, food, etc.) by Tamarin et al. [47]. They grafted a monoclonal antibody (AM13 MAb) against M13 bacteriophage on the device surface (SiO2) and sensed the M13 bacteriophage/AM13 immunoreaction. The authors suggested the potentialities of such acoustic biosensors for biological detection. The same year, it was shown that mass sensitivity of LW devices with ZnO layer was larger than that of sensors with SiO2 guiding layers [48]. The authors of this work monitored adsorption of rat immunoglobulin G, obtain‐ ing mass sensitivities as high as 950 cm2 /g. They pointed out that such a device was a prom‐ ising candidate for immunosensing applications.

An aptamer-based LW sensor which allowed the detection of small molecules was devel‐ oped by Schlensog et al. in 2004 [81]. This biosensor offers an advantage over immunosen‐ sors, since it does not require the production of antibodies against toxic substances. A LW biosensor for the detection of pathogenic spores at or below inhalational infectious levels was reported by Branch et al. in 2004 [20]. A monoclonal antibody with a high degree of se‐ lectivity for anthrax spores was used to capture the non-pathogenic simulant *Bacillus thurin‐ giensis* B8 spores in aqueous conditions. The authors stated that acoustic LW biosensors will have widespread application for whole-cell pathogen detection.

Moll et al. developed an innovative method for the detection of *E. coli* employing an LW de‐ vice in 2007 [49]; it consisted of grafting goat anti-mouse antibodies (GAM) onto the sensor surface and introducing *E. coli* bacteria mixed with anti-*E. coli* MAb in a second step. The sensor response time was shorter when working at 37°C, providing results in less than 1 hour with a detection threshold of 106 bacteria/mL. More recently, the same group described a multipurpose LW immunosensor for the detection of bacteria, virus and proteins [82]. They successfully detected bacteriophages and proteins down to 4 ng/mm2 and *E.coli* bacte‐ ria up to 5.0 × 105 cells in a 500 µL chamber, with good specificity and reproducibility. The authors stated that whole bacteria can be detected in less than one hour.

Andrä et al. used a LW sensor to investigate the mode of action and the lipid specificity of human antimicrobial peptides [83]. They analyzed the interaction of those peptides with model membranes. These membranes, when attached to the sensor surface, mimic the cyto‐ plasmic and the outer bacterial membrane. A LW immunosensor was used in 2008 by Bisoffi et al. [84] to detect Coxsackie virus B4 and Sin Nombre Virus (SNV), a member of the hanta‐ virus family. They described a robust biosensor that combines the sensitivity of SAW at a frequency of 325 MHz with the specificity provided by monoclonal and recombinant anti‐ bodies for the detection of viral agents. Rapid detection (within seconds) for increasing virus concentrations was reported. The biosensor was able to detect SNV at doses lower than the load of virus typically found in a human patient suffering from hantavirus cardiopulmonary syndrome.

In 2009, it was shown the possibility to graft streptavidin-gold molecules onto a LW sensor surface in a controlled way and was demonstrated the capability of the sensor to detect nano-particles in aqueous media by Fissi et al. [85]. In 2010, a complementary metal–oxide semiconductor CMOS-LW biosensor for breast cancer biomarker detection was presented by Tigli et al. [36]. This biosensor was fabricated using CMOS technology and used gold as guiding layer and as interface material between the biological sensing medium and the transducer.

LW devices were used as sensors for okadaic acid immono-detection through immobilized specific antibodies by Fournel et al. [76]. They obtained three times higher frequency shifts with the okadaic acid than with an irrelevant peptide control line. A LW based bacterial bio‐ sensor for the detection of heavy metal in liquid media was reported in 2011 by Gammoudi et al. [86]. Whole bacteria (*E. coli*) were fixed as bioreceptors onto the acoustic path of the sensor coated with a polyelectrolyte multilayer using a layer by layer electrostatic self-as‐ sembly procedure. Changes of bacteria viscoelastic equivalent parameters in presence of toxic heavy metals were monitorized.

A LW-based wireless biosensor for the simultaneous detection of Anti- Dinitrophenyl-KLH (anti-DNP) immunoglobulin G (IgG) was presented by Song et al. in 2011 [87]. They used poly(methyl-methacrylate) (PMMA) guiding layer and two sensitive films (Cr/Au). A LW sensor whose phase shifts as a function of the immobilized antibody quantity, combined with an active acoustic mixing device, was proposed by Kardous et al. [88] in 2011. They as‐ sessed that mixing at the droplet level increases antibodies transfer to a sensing area surface and increases the reaction kinetics by removing the dependency with the protein diffusion coefficient in a liquid, while inducing minimum disturbance to the sensing capability of the Love mode. LW sensors have been also used to study the properties of protein layers [40], DNA [89,90] and detect the adsorption and desorption of a lipid layer [91].

Currently, the only commercial LW biosensor system available in the market is commercial‐ ized by the German company *SAW instruments GmbH*. The sensor system can achieve a limit of detection (LOF) of 0.05 ng/cm2 with a sample volume of 40-80 µL. *Senseor* company (Mou‐ gins, France) has a commercially available microbalance development kit (SAW-MDK1) which consists of a two-channel LW delay lines.

## **8. Trends and future challenges of LW sensors**

LW biosensors have not been very well recognized by the scientific community [72] nor by the market yet. This might be due to the technological hindrances found for applying this device as biosensor, since it is sensitive to changes in the viscoelastic properties of the coat‐ ing, which complicates the results interpretation. Reports about applications where mass al‐ terations are separated from viscoelastic effects can enhance the acceptance of LW sensors [72]. Hence, it is necessary to investigate different alternatives for carrying out the procedure of parameters extraction with LW sensors. Nowadays, the trend is the placement of multi‐ ple, small, versatile sensors into a network configured for a specific location [10]. LW devi‐ ces will move into the lab-on-a chip arena during the next years. Nevertheless, LW devices still have some hurdles to clear. LW biosensors packaging needs further development and cost reduction. In addition, much research and efforts are still required addressing the fluid‐ ic technology issue. Integration and automation with electronics and flow cells reduce costs of the system and increase the throughput. For a better performance of LW sensors, the com‐ bination with other detection methods such as optical [32] or chromatographic [92] are being considered.

Mathematical modeling and simulations of these devices are also essential for the develop‐ ment of new sensors, especially with respect to the study of new materials and wave propa‐ gation [72]. Numerical calculations and FEM analysis of LW sensors could help for further understanding of these devices.
