Designing a Non-invasive Surface Acoustic Resonator for Ultra-high Sensitive Ethanol Detection for an On-the-spot Health Monitoring System

Surface acoustic wave (SAW) sensors–based on piezoelectric crystal resonators–are extremely sensitive to even very small perturbations in the external atmosphere, because the energy associated with the acoustic waves is confined to the crystal surface. In this study, we present a critical review of the recent researches and developments predominantly used for SAW-based organic vapor sensors, especially ethanol. Besides highlighting their potential to realize real-time ethanol sensing, their drawbacks such as indirect sensing, invasive, time initializing, and low reliability, are properly discussed. The study investigates a proposed YZ-lithium niobate piezoelectric substrate with interdigital transducers patterned on the surface. Design of the resonator plays an important role in improving mass sensitivity, particularly the sensing area. Accordingly, a tin dioxide (SnO₂) layer with a specific thickness is generated on the surface of the sensor because of its high affinity to ethanol molecules. To determine the values of sensor configuration without facing the practical problems and the long theoretical calculation time, it is shown that the mass sensitivity of SAW sensors can be calculated by a simple three-dimensional (3-D) finite element analysis (FEA) using a commercial finite-element platform. In design validation step, different concentrations of ethanol are applied to investigate the acoustic wave properties of the sensor. The FEA data are used to obtain the surface and bulk total displacements of the sensor and fast Fourier transform (FFT) on output spectrum. The sensor could develop into highly sensitive and fast responsive structure so that a positive intensity shift of 0.18e-2 RIU is observed when the sensor is exposed to 15 ppm ethanol. It is capable of continuously monitoring the ethanol gas whether as an ultra-high sensitive sensor or switching applications for medical and industrial purposes.     

Simultaneous stochastic sensing of divalent metal ions

Stochastic sensing is an emerging analytical technique that relies upon single-molecule detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concentration of an analyte and, through a characteristic signature, its identity. Here, we show that the concentrations of two or more divalent metal ions in solution can be determined simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.     

Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1mg/ml BSA (blocking agent) onto the magnetoelastic resonator's surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.     

Covalent bound sensing layers on surface acoustic wave (SAW) biosensors

This paper reports on the development of immunosensors based on commercially available surface acoustic wave (SAW) devices working at 380 MHz. Approaches for coating the sensor surface with a sensing layer of receptive biomolecules are presented and discussed. It was found that the sensitivity strongly relates to the immobilization method. Additionally, the sensitivity can be influenced by the density of accessible biomolecules on the active sensing area. Usually, by most of the standard immobilization procedures, two-dimensional layers of receptive biomolecules are obtained. We present a three-dimensional layer, which provides a higher absolute amount of recognition molecules. A dextran layer is photoimmobilized to the sensor surface and the recognition molecules are covalently embedded into the dextran matrix. The feasibility of specific immunosensing is investigated using SAW sensors connected to a fluid handling system.     

Gas phase activity of anti-FITC antibodies immobilized on a surface acoustic wave resonator device

In this paper we present the results of a series of experiments on the activity of antibodies in a vapor phase sensor. For these experiments the sensor component was a ST-Quartz resonator with a center frequency of approximately 250 MHz. Anti-FITC antibodies were attached to the electrodes on the device surface via a protein-A crosslinker. Surface acoustic wave (SAW) resonator devices with various coatings were mounted in TO-8 packages, inserted into our sensor head module and subjected to various fluorescent analyte gases. Numerous controls were performed including the use of coated and uncoated devices along with devices coated with antibodies which were not specific for the target analyte. The SAW immunosensor response was monitored and a baseline frequency shift was observed when the analyte being presented was the antigen for the immobilized antibody. To provide an independent measure of antibody/antigen binding, the devices were removed from the sensor head, washed with a buffer solution to remove any unbound analyte, and then inspected using a confocal laser scanning microscope (CLSM). Since all the analytes being used in these experiments were fluorescent this afforded us the opportunity to visualize the attachment of the analyte to the antibody film. Given the high resolution of the CLSM, we were able to identify the location of the attachment of the fluorescent analytes relative to the 1.5 microm wide electrodes of the SAW device. We believe that these experiments demonstrate that we have achieved real time molecular recognition of these small molecules in the vapor phase.     

A surface plasmon resonance immunosensor based on the streptavidin-biotin complex.

It is shown that a streptavidin monolayer immobilized onto an evaporated gold film with biotin forms the basis of a highly specific sensing element. As an example, we show that by immobilizing the biotinylated antibody sex hormone binding globulin (alpha-SHBG) to the bound streptavidin monolayer a specific sensor for the antigen SHBG is readily fabricated. The interaction between immobilized antibody and corresponding antigen is monitored by surface plasmon resonance spectroscopy and is shown to follow a classic Langmuir isotherm. Detection of SHBG at nanomolar concentrations is demonstrated.     

Immobilization of pneumococcal polysaccharide vaccine on silicon oxide wafer for an acoustical biosensor.

One the most important aspects of a biosensor is related to immobilization and maintenance of specific reference compounds on sensing surfaces. A method for the immobilization of polysaccharides to a silicon oxide surface intended for Surface Acoustical Waves (SAW) sensors is described. Silicon oxide is a hydrophobic inorganic support used for the fabrication of many electronic devices. The pneumococcal polysaccharide (PPS) vaccine is immobilized via Protein A after pre-treatment of the surface with hydrochloric acid. The effects of non-specific binding are discussed. The results indicate that the immobilization of PPS via Protein A increases the sensitivity of detecting Streptococcus pneumoniae antibodies in human sera and offers greater reproducibility of response compared with ELISA methods. The principles of this technique are simple and are applicable to the immobilization of many capsular polysaccharides.     

SPR biosensing coupled to a digital microfluidic microstreaming system

This article reports on a proof-of-concept system composed of a droplet based surface plasmon resonance (SPR) system coupled to a surface acoustic wave (SAW) microfluidic plateform. It is now well established that surface based binding analyses such as SPR are highly influenced by the transport of analyte to the sensing surface. Further, obtaining reliable equilibrium in flow cells to realize quantification studies is not straightforward. An original solution compared to generally used pressure driven flows is then proposed to favourably cope with these issues. Efficiency of SAW microstreaming coupled to SPR biosensing is considered, in order to improve the accuracy of kinetic parameter estimation in mass transport limited regime and to realize reliable quantification studies. First, the droplet based SPR technique and its advantages are presented. Then, the integration of the microstreaming on the system is discussed. Streptavidin binding is then monitored in static mode and under SAW streaming mode.     

Magnetic acoustic resonance immunoassay (MARIA): a multifrequency acoustic approach for the non-labelled detection of biomolecular interactions

A unique sensing platform, comprising an electromagnetic field detector and an acoustic resonator, has been used as a wireless system for remote sensing of biorecognition events. The MARS (Magnetic Acoustic Resonator Sensor) technique has proven useful for detecting the formation of protein multilayers derived from specific binding phenomena. The technique enables multifrequency analysis, without the need of electrodes attached to the sensing element, and also facilitates the in situ surface modification of the substrate for antibody attachment. The MARS sensor was utilized as the platform on which a standard immunoassay was carried out. Two different conditions for the attachment of the first antibody to the quartz surface were tested: (i) Adsorption of the antibody onto the surface of a bare quartz disc; (ii) covalent immobilization of the antibody to a chemically modified quartz surface. Both methods can be successfully utilized for the 'label-less' detection of the biorecognition event between goat IgG and anti-goat IgG by analysis of the multifrequency spectrum. Covalent attachment of the primary antibody results in a more efficient immobilization, with higher surface density, and a consistently enhanced response for the binding of the secondary antibody. This approach will be of interest to life scientists and biochemists that require high performance assay methodologies that do not use chemical labels.     

Kinetics parameter estimation for the binding process of salicylic acid to human serum albumin (HSA) with capacitive sensing technique

A novel method for real-time investigating the binding interaction between human serum albumin (HSA) and salicylic acid with capacitive sensing technique was successfully proposed. HSA was immobilized on the surface of a gold electrode modified with an insulating poly (o-phenylenediamine) (o-PD) film and colloid Au nanoparticles layers. The bioactivity of HSA was remained and major binding sites were available because of the excellent biocompatibility of gold nanoparticles. The capacitance and interfacial electron resistance of the sensor were altered, owing to the binding of HSA to salicylic acid. The time courses of the capacitance change were acquired with capacitive sensing technique during the binding process. Based on the capacitance response curves with time, the response model for the binding was derived in theory and the corresponding regression parameters were determined by fitting the real-time experimental data to the model. The binding and the dissociation rate constants (k(1) and k(-1)) were estimated to be 54.8 (mol l(-1))(-1) s(-1) and 2.9 x 10(-3) s(-1), respectively. And the binding equilibrium constant (K(a)) was calculated to be 1.89 x 10(4) (mol l(-1))(-1).     

A Review on Functionalized Gold Nanoparticles for Biosensing Applications

Nanoparticle technology plays a key role in providing opportunities and possibilities for the development of new generation of sensing tools. The targeted sensing of selective biomolecules using functionalized gold nanoparticles (Au NPs) has become a major research thrust in the last decade. Au NP-based sensors are expected to change the very foundations of sensing and detecting biomolecules. In this review, we will discuss the use of surface functionalized Au NPs for smart sensor fabrication leading to detection of specific biomolecules and heavy metal ions.     

Influence of intermediate aminodextran layers on the signal response of surface acoustic wave biosensors

Surface acoustic wave (SAW) devices based on horizontally polarized surface shear waves enable direct and label-free detection of proteins in real time. Binding reactions on the sensor surface are detected by determining changes in surface wave velocity caused mainly by mass adsorption or change of viscoelasticity in the sensing layer. Intermediate hydrogel layers have been proven to be useful to immobilize capture molecules or ligands corresponding to the analyte. However, the SAW signal response strongly depends on the morphology of the hydrogel due to different relative changes of its acoustomechanical parameters such as viscoelasticity and density. In this work five aminodextrans (AMD) and one diamino polyethylene glycol (DA-PEG) were used as intermediate hydrogel layers. Sensors with immobilized streptavidin and samples containing biotinylated bovine serum albumin were used to exemplify affinity assays based on immobilized capture molecules for protein detection. The effects of the three-dimensional AMDs and the two-dimensional (2D) DA-PEG on the SAW signal response were investigated. The signal height decreased with increasing molar mass and increasing amount of immobilized AMD. Consequently, thin hydrogel layers are ideal to obtain optimum signal responses in this type of assay, whereas it is not necessarily a 2D hydrogel that gives the best results.     

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.     

An interleukin-6 ZnO/SiO(2)/Si surface acoustic wave biosensor

A novel high sensitivity ZnO/SiO(2)/Si Love mode surface acoustic wave (SAW) biosensor for the detection of interleukin-6 (IL-6), is reported. The biosensors operating at 747.7MHz and 1.586GHz were functionalized by immobilizing the monoclonal IL-6 antibody onto the ZnO biosensor surface both through direct surface adsorption and through covalent binding on gluteraldehyde. The morphology of the IL-6 antibody-protein complex was studied using scanning electron microscopy (SEM), and the mass of the IL-6 protein immobilized on the surface was measured from the frequency shift of the SAW resonator biosensor. The biosensor was shown to have extended linearity, which was observed to improve with higher sensor frequency and for IL-6 immobilization through the monoclonal antibody. Preliminary results of biosensor measurements of low levels of IL-6 in normal human serum are reported. The biosensor can be fully integrated with CMOS Si chips and developed as a portable real time detection system for the interleukin family of proteins in human serum.     

Binding kinetics of grouper nervous necrosis viruses with functionalized antimicrobial peptides by nanomechanical detection

We report the binding kinetics of fish-infected grouper nervous necrosis viruses (NNV) and selected antimicrobial peptides (AMPs) by nanomechanical detection. AMPs, the vital member in an innate immunity, are promising candidates in the fight against pathogens due to their broad range of antimicrobial activity and low toxicity. Grouper NNV primarily cause mass mortality of many marine cultured fish species, and two selected AMPs in this study were found to inhibit viruses by agglutinating its virions to form aggregates. The binding activity of NNVs with functionalized AMPs onto a sensing microcantilever yielded induced surface stresses, indicating high binding strength of molecular interaction. The binding affinity and kinetic rate constants of molecular recognition events calculated for NNV-AMP(TH1-5) compared to NNV-AMP(cSALF) were found to be 2.1-fold and 4.43-fold, respectively, indicating TH1-5 effectively bind with NNV more than cSALF. Moreover, a microscopic X-ray photoelectron spectroscopy technique was employed for further validation of pre- and post-NNV binding onto peptides-functionalized sensing surface. An increase in the spectrum and intensity of the P 2p and N 1s elements for the post-NNV binding was clearly shown to ensure the existence of phosphate groups and nitrogen-containing ring structures of specific NNV-TH1-5 interaction. Therefore, the microcantilever biosensing technique provides a potential and useful screening of AMPs for affinity to NNVs.     

Detection of Escherichia coli O157:H7 with langasite pure shear horizontal surface acoustic wave sensors

The toxigenic Escherichia coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure and death. On average, O157:H7 causes 73,000 illnesses, 2100 hospitalizations and 60 deaths annually in the United States alone. There is the need for sensors capable of rapidly detecting dangerous microbes in food and water supplies to limit the exposure of human and animal populations. Previous work by the authors used shear horizontal surface acoustic wave (SH SAW) devices fabricated on langasite (LGS) Euler angles (0°, 22°, 90°) to successfully detect macromolecular protein assemblies. The devices also demonstrated favorable temperature stability, biocompatibility and low attenuation in liquid environments, suggesting their applicability to bacterial detection. In this paper, a biosensor test setup utilizing a small volume fluid injection system, stable temperature control and high frequency phase measurement was applied to validate LGS SH SAW biosensors for bacterial detection. The LGS SH SAW delay lines were fabricated and derivatized with a rabbit polyclonal IgG antibody, which selectively binds to E. coli O157:H7, in this case a non-toxigenic test strain. To quantify the effect of non-specific binding (negative control), an antibody directed against the trinitrophenyl hapten (TNP) was used as a binding layer. Test E. coli bacteria were cultured, fixed with formaldehyde, stained with cell-permeant nucleic acid stain, suspended in phosphate buffered saline and applied to the antibody-coated sensing surfaces. The biosensor transmission coefficient phase was monitored using a network analyzer. Phase responses of about 14° were measured for the E. coli detection, as compared to 2° due to non-specific anti-TNP binding. A 30:1 preference for E. coli binding to the anti-O157:H7 layer when compared to the anti-TNP layer was observed with fluorescence microscopy, thus confirming the selectivity of the antibody surface to E. coli.     

Combination of a SAW-biosensor with MALDI mass spectrometric analysis

A S-sens K5 surface acoustic wave biosensor was coupled with mass spectrometry (SAW-MS) for the analysis of a protein complex consisting of human blood clotting cascade factor alpha-thrombin and human antithrombin III, a specific blood plasma inhibitor of thrombin. Specific binding of antithrombin III to thrombin was recorded as a function of time with a S-sens K5 biosensor. Two out of five elements of the sensor chip were used as references. To the remaining three elements coated with RNA anti-thrombin aptamers, thrombin and antithrombin III were bound consecutively. The biosensor measures mass changes on the chip surface showing that 20% of about 400fmol/cm2 thrombin formed a complex with the 1.7-times larger antithrombin III. Mass spectrometry (MS) was applied to identify the bound proteins. Sensor chips with aptamer-captured (1) thrombin and (2) thrombin-antithrombin III complex (TAT-complex) were digested with proteases on the sensor element and subsequently identified by peptide mass fingerprint (PMF) with matrix assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry. A significant identification of thrombin was achieved by measuring the entire digest with MALDI-ToF MS directly from the sensor chip surface. For the significant identification of both proteins in the TAT-complex, the proteolytic peptides had to be separated by nano-capillary-HPLC prior to MALDI-ToF MS. SAW-MS is applicable to protein interaction analysis as in functional proteomics and to miniaturized diagnostics.     

Recent advances in the development of bioelectronic nose

The olfactory system has the ability to discriminate and identify thousands of odorant compounds at very low concentrations. Recently, many researchers have been trying to develop artificial sensing devices that are based on the olfactory system. A bioelectronic nose, which uses olfactory receptors (ORs) as sensing elements, would benefit naturally optimized molecular recognition. Accordingly, ORs can be effectively used as a biological element in bioelectronic noses. Bioelectronic nose can be classified into cell-based and protein-based biosensors. The cell-based biosensor uses living cells that express olfactory receptors as the biological sensing elements and the protein-based biosensor uses the olfactory receptor protein. The binding of odorant molecules to the ORs can be measured using various methods such as piezoelectric, optic, and electric devices. Thus, bioelectronic nose can be developed by combining the biological sensing elements with these non-biological devices. The application of bioelectronic nose in a wide range of different scientific and medical fields is essentially dependent on the development of highly sensitive and selective biosensors. These sensor systems for the rapid detection of specific odorants are crucial for environmental monitoring, anti-bioterrorism, disease diagnostics, and food safety. In this article, we reviewed recent advances in the development of bioelectronic nose.     

Label-free THz sensing of living body-related molecular binding using a metallic mesh

We have demonstrated label-free THz sensing of living body-related molecular binding using a thin metallic mesh and a polyvinylidene difluoride (PVDF) membrane. Metallic meshes in the THz region are designed for anomalous transmission phenomena derived from a resonant excitation of surface waves. Additionally, they are designed to have a sharp dip in transmittance. The metallic mesh is very sensitive to a change of the refractive index of materials attached to the metallic mesh. In this paper, we report sensing of interactions between lectin and sugar using this technique. We found that the dip frequency shift, transmittance attenuation of the dip frequency, and peak shift of the derivative spectrum of the phase shift depend on the bonding amount of lectin–sugar interactions. We also applied this technique to detect avidin–biotin interactions, leading to the detection of a small amount of biotin (0.17 pg/mm²).     

Multi-analyte surface plasmon resonance biosensing

Surface plasmon resonance (SPR) biosensors are affinity sensing devices exploiting a special mode of electromagnetic field-surface plasmon-polariton-to detect the binding of analyte molecules from a liquid sample to biomolecular recognition elements immobilized on the surface of the sensor. In this paper, we review advances of SPR biosensor technology towards detection systems for the simultaneous detection of multiple analytes (multi-analyte detection). In addition, we report application of a recently developed multichannel SPR sensor based on spectroscopy of surface plasmons and wavelength division multiplexing of sensing channels to multi-analyte detection.