Microbalance Biosensor Research Articles

Electric Field-Assisted Molecularly Imprinted Polymer-Modified QCM Sensor for Enhanced Detection of Immunoglobulin.

In this study, an electric-field-assisted molecularly imprinted polymer (EFAMIP) as an enhanced form of MIP was developed to improve the MIP-modified quartz crystal microbalance (QCM) biosensors. While exerting a vertical electric field, polymerization of methacrylic acid in the presence of immunoglobulin G (IgG) as the template was initiated, and later, after the template removal process, the EFAMIPs were obtained. The polymer surface characterization was conducted by using a scanning electron microscope. The impact of electric field direction on IgG binding sites, forming either EFAMIP-Fab or EFAMIP-Fc, was assessed. Next, the static measurement results in liquid for EFAMIP-modified QCM and MIP-modified QCM were compared. While encompassing IgG, EFAMIP-modified QCMs exhibited up to a 113.5% higher frequency shift than typical MIP in time-limited detection. The final frequency shift of EFAMIP, which determines the detection limit of IgG, was improved up to 12.5% compared to typical MIP. Moreover, the EFAMIP-Fab performance was promising for the selective detection of IgG in a solution containing different types of immunoglobulins.

Design, Fabrication and Performance Analysis of a Portable, Antenna Analyzer Based, Quartz Crystal Microbalance Measuring System with Energy Dissipation

Impedance measurements play a critical role in analyzing the electrical behavior of piezoelectric biosensors in general. Antenna analyzers are engineered to measure the specific case of input impedance for antenna systems. In this study small form factor antenna analyzer is repurposed to work as driving circuit for a Quartz Crystal Microbalance (QCM) biosensor in combination with a single board computer as an indication of how small and portable an impedance measuring system can be made, while allowing monitoring of important parameters of series and parallel resonance frequencies together with dissipation factor. A QCM crystal with a 10 MHz fundamental resonance frequency is employed to determine the limit of detection of the system in Bovine Serum Albumin (BSA) and glycerol solutions. Dissipation factor and phase angle were monitored during the experiments. Limit of detection is 20 µg/ml BSA in phosphate buffer saline (PBS) and 250 µl of glycerol in 100 ml of deionized water.

Development of gold nanospikes-modified quartz crystal microbalance biosensor for prostate specific antigen detection

Prostate-specific antigen (PSA) screening is vital for the early detection and assessment of prostate cancer treatment efficacy. Integrating nanotechnology into biosensor development allows for targeted biomarker detection. In this study, we present the fabrication of gold nanospikes (AuNS) structures on a quartz crystal microbalance (QCM) using the pulse current electrodeposition (PCD) technique. By varying the duty cycles of PCD, we achieved the desired nanostructure. The characteristics of the AuNS-modified QCM electrode were thoroughly investigated using various micro-structural characterization techniques such as x-ray diffraction (XRD), field emission scanning electron microscope (FESEM), atomic force microscope (AFM), X-ray photoelectron spectroscopy (XPS) and cyclic voltammetry (CV). Prior to biomolecule immobilization, each chemical treatment was verified through contact angle and FT-IR analyses. The performance of the developed QCM biosensor was evaluated using PSA at various concentrations. The results revealed that the frequency variation of the QCM biosensor was directly proportional to the PSA concentration. Notably, the AuNS-modified QCM biosensor exhibited the highest frequency shift at a 20 % duty cycle, demonstrating a linear detection range of 0.1–100 ng mL−1 and a limit of detection (LOD) of 24 pg mL−1. The developed QCM biosensor significantly enhances detection efficiency by increasing the number of binding sites for biomolecules through an enlarged surface area. The demonstrated sensitivity of the AuNS-modified QCM biosensor indicates its potential to address the increasing demand for early diagnosis and screening in healthcare systems. This innovative biosensor technology holds promise for improving prostate cancer detection and has broader implications for the development of highly sensitive biosensors for various biomarkers.

MEMS‐Based Ultra‐High Frequency Wireless 10 × 10 QCM Sensor Array for Biochemical Sensing Applications

The quartz crystal microbalance (QCM) biosensor is one of the devices that enables label‐free evaluation of biomolecular reactions. Commercial QCM biosensors have less than 10 channels for detection and are not suitable for the screening. The sensitivity of the QCM biosensor increases as the quartz thickness becomes thinner; however, the limit of thinning is 60–330 μm. In this study, applying micromachining technology, a QCM sensor array chip of 15 mm square that had 100 ultra‐thin quartz resonators (2.5 μm) that were operated wirelessly was developed. And we succeeded in detecting the resonance spectrum through the device evaluation. © 2024 Institute of Electrical Engineers of Japan. Published by Wiley Periodicals LLC.

Coverage Effects in Quartz Crystal Microbalance Measurements with Suspended and Adsorbed Nanoparticles.

Quartz crystal microbalance (QCM) biosensors often deal with nanoparticles suspended in the solvent at tens of nanometers above the resonator while being linked to some molecular receptor (DNA, antibody, etc.). This work presents a numerical analysis based on the immersed boundary method for the flow and QCM impedance created by an ensemble of spherical particles of radius R at varying surface coverage Θ and particle-surface gap distance Δ. The trends for the frequency Δf and dissipation ΔD shifts against Θ and Δ are shown to be determined by modifications in the structure of the perturbative flow created by the analytes. Simulations are in good agreement with a relatively large experimental database collected from the literature. Qualitative differences between the adsorbed (Δ ≈ 0) and suspended states (Δ > 0) are highlighted. In the case of adsorbed particles, deviations from the linear scaling Δf ∝ Θ are observed above Θ > 0.05 and largely depend on the specific analyte-substrate combination. Moreover, in general, ΔD(Θ) is not monotonous and usually presents a maximum around Θ ∼ 0.2. In the case of suspended analytes, the agreement with the numerical results is quantitative, indicating that the predicted scalings are universal and determined by hydrodynamics. Up to high coverage, the suspended particles present Δf ∼ Θ and ΔD ∼ Θβ, where β ≈ 0.85 is not largely dependent on R. The present findings should help forecast molecular configurations from QCM signals and have implications on QCM analyses, e.g., in the case of suspended ligands (Δf ∝ Θ), it is safe to use Δf to build Langmuir isotherms and estimate equilibrium constants. Open questions on the transition from the suspended-to-adsorbed state are discussed.

Sugar-Lectin Interactions for Direct and Selective Detection of Escherichia coli Bacteria Using QCM Biosensor.

Pathogenic Escherichia coli (E. coli) remains a safety concern in the preservation and quality of green leafy vegetables. Sugar-lectin interactions provide a reliable, specific, and effective sensing platform for the detection of bacteria as compared to the tedious conventional plate counting technique. Herein, we present the synthesis of 4-(N-mannosyl) benzoic acid (4-NMBA) and 4-thiophenyl-N-mannose (4-TNM) via a two-step reductive amination for the detection of E. coli using a quartz crystal microbalance (QCM) biosensor. The 4-NMBA was synthesized with mannose and para-aminobenzoic (4-PBA), while the 4-TNM was synthesized with mannose and 4-aminophenyl disulfide (4-AHP) using water and acetic acid in a 1:1 ratio. The resultant structure of mannose derivatives (4-NMBA and 4-TNM) was characterized and confirmed using analytical tools, such as Mass Spectrometer, SEM, and FTIR. The choice of ligands (mannose derivatives) is ascribed to the specific recognition of mannose to the FimH lectin of the type 1 pilus of E. coli. Furthermore, the 4-PBA and 4-AHP conjugated to mannose increase the ligand affinity to FimH lectins. The setup of the QCM biosensor was composed of modification of the crystal surface and the covalent attachment of ligands for the detection of E. coli. The piezoelectric effect (frequency shift of the quartz) was proportional to the change in mass added to the gold crystal surface. Both the 4-NMBA- and 4-TNM-coated QCM sensors had a limit of detection of 3.7 CFU/mL and 6.6 CFU/mL with a sensitivity of 2.56 × 103 ng/mL and 8.99 × 10-5 ng/mL, respectively, within the dynamic range of 103 to 106 CFU/mL. This study demonstrates the application of ligand-coated QCM biosensors as a cost-effective, simple, and label-free technology for monitoring pathogenic bacteria via molecular interactions on crystal surfaces.

Development of a quartz crystal microbalance-based immunosensor for the early detection of mesothelin in cancer

A robust real-time flow injection assay using Quartz Crystal Microbalance (QCM) biosensor has been developed for the detection of mesothelin, an antigen expressed on various malignant tumors including mesothelioma and ovarian cancers. A QCM sensor chip functionalized with self-assembled monolayer of cysteamine used to fabricate a sensitive immunosensor. Mesothelin specific antibody was immobilized on cysteamine modified gold surface of quartz crystal using N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide and N-hydroxy succinimide coupling. Further, ethanolamine was used as a blocking reagent to prevent the non-specific adsorption on antibody functionalized gold surface. Various concentrations of mesothelin was tested and the resonant frequency variation of the crystal was observed by a quartz crystal microbalance until a stable response is obtained. A good correlation between the frequency changes and concentration of mesothelin tested was observed and the QCM biosensor detects mesothelin in the linear range of 100pg/mLto 50 ng/mL. Thus, QCM assay could be a promising technique for early diagnosis of mesothelioma, ovarian and pancreatic adenocarcinoma.

Detection of Salmonella Typhimurium with Gold Nanoparticles Using Quartz Crystal Microbalance Biosensor.

Demonstration of the Salmonella Typhimurium detection system was shown utilizing a quartz crystal microbalance (QCM) biosensor and signal enhancement by gold nanoparticles. In this study, a benchtop system of a QCM biosensor was utilized for the detection of Salmonella Typhimurium. It was designed with a peristaltic pump system to achieve immobilization of antibodies, detection of Salmonella, and the addition of gold nanoparticles to the sensor. As a series of biochemical solutions were introduced to the surface, the proposed system was able to track the changes in the resonant frequency which were proportional to the variations of mass on the sensor. For antibody immobilization, polyclonal antibodies were immobilized via self-assembled monolayers to detect Salmonella O-antigen. Subsequently, Salmonella Typhimurium was detected by antibodies and the average frequency before and after detecting Salmonella was compared. The highest frequency shifts were -26.91 Hz for 109 CFU/mL while the smallest frequency shift was -3.65 Hz corresponding to 103 CFU/mL. For the specificity tests, non-Salmonella samples such as E. coli, Listeria, and Staphylococcus resulted in low cross-reactivity. For signal amplification, biotinylated antibodies reacted to Salmonella followed by streptavidin-100 nm AuNPs through biotin-avidin interaction. The frequency shifts of 103 CFU/mL showed -28.04 Hz, and consequently improved the limit of detection.

A quartz crystal microbalance biosensor based on polyethylenimine-modified gold electrode to detect hepatitis B biomarker

Biomarkers-based QCM-biosensors are suitable tools for the label-free detection of infectious diseases. In the current study, a QCM-biosensor was developed for the detection of HBsAg. Briefly, anti-HBsAg antibodies were covalently bound to the primary amines after PEI and thiolated-PEI surface modifications of gold-electrode. After RSM optimization, the statistical analysis revealed no significant difference between the immobilization yields of modified layers. Therefore, the PEI-modified QCM-biosensor was selected for further analysis. The PEI-surface was evaluated by FESEM, AFM, ATR-FTIR, and CA measurement. The surface hydrophilicity and its roughness were increased after PEI-coating. Also, FTIR confirmed the PEI-layering on the gold-surface. RSM optimization increased the antibody immobilization yield up to 80%. The QCM-biosensor showed noteworthy results with a wide dynamic range of 1–1 × 103 ng/mL, LOD of 3.14 ng/mL, LOQ of 9.52 ng/mL, and detection capability in human-sera, which were comparable with the ELISA. The mean accuracy of the QCM-biosensor was obtained at 91% when measured by the spike recovery test using human-sera. The biosensor was completely regenerated using 50 mM NaOH and 1% SDS. The benefits provided by the developed biosensor such as broad dynamic range, sensitivity, selectivity, stability, regenerate ability, and low cost suggest its potential application for the non-invasive and timely monitoring of HBV-biomarker.

Protein adsorption on amino-acid-conjugated self-assembled molecule-modified SiO2 surfaces

Protein adsorption has a crucial effect on biocompatibility during the interaction of biomaterial surfaces and the biological environment. It is significant to understand and control the interactions among biomaterials and proteins for several biomedical applications. Surface engineering plays a significant role in determining biocompatibility by tuning the effects directly on proteins. In this study, amino acid (histidine and leucine) conjugated self-assembled molecules were synthesized and used to modify silicon dioxide (SiO2) surfaces to investigate protein adsorption behavior. Silicon dioxide surfaces were modified with (3-aminopropyl)triethoxysilane-conjugated histidine and leucine amino acids. Modified silicon dioxide surfaces were characterized by water contact angle measurements and X-ray photoelectron spectroscopy analysis. Protein adsorption (human serum albumin, fibrinogen and immunoglobulin G) on silicon dioxide-coated crystals was investigated in situ by using a quartz crystal microbalance (QCM) biosensor. From the results, model proteins showed different selectivities to the amino-acid-conjugated silicon dioxide-coated crystals depending on the type of the amino acid and concentration. Consequently, this controlled chemistry on the surface of biomaterials has a great potential to manipulate protein adsorption and enhance the biocompatibility of biomaterials for various biomedical applications.

Recent Advances in Quartz Crystal Microbalance Biosensors Based on the Molecular Imprinting Technique for Disease-Related Biomarkers

The molecular imprinting technique is a quickly developing field of interest regarding the synthesis of artificial recognition elements that enable the specific determination of target molecule/analyte from a matrix. Recently, these smart materials can be successfully applied to biomolecule detection in biomimetic biosensors. These biosensors contain a biorecognition element (a bioreceptor) and a transducer, like their biosensor analogs. Here, the basic difference is that molecular imprinting-based biosensors use a synthetic recognition element. Molecular imprinting polymers used as the artificial recognition elements in biosensor platforms are complementary in shape, size, specific binding sites, and functionality to their template analytes. Recent progress in biomolecular recognition has supplied extra diagnostic and treatment methods for various diseases. Cost-effective, more robust, and high-throughput assays are needed for monitoring biomarkers in clinical settings. Quartz crystal microbalance (QCM) biosensors are promising tools for the real-time and quick detection of biomolecules in the past two decades A quick, simple-to-use, and cheap biomarkers detection technology based on biosensors has been developed. This critical review presents current applications in molecular imprinting-based quartz crystal microbalance biosensors for the quantification of biomarkers for disease monitoring and diagnostic results.

Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50–80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography – asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV – ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.

Machine learning approach to optimization of parameters for impedance measurements of Quartz Crystal Microbalance to improve limit of detection

Impedance spectroscopy allows real time monitoring of fundamental resonance frequency and harmonics of AT cut Quartz Crystal Microbalance (QCM) biosensors. Acquisition of accurate and high resolution spectroscopic data requires optimization of parameters, which is rarely performed as it requires an excessive number of experiments. In this work, machine learning clustering and classification algorithms are applied in an attempt to minimize the number of experiments by designating a small representative subset of chosen values of parameters, while preserving the variation of those values. Our results indicated it is possible to reduce the number of experiments required by more than 10 fold. The constructed model has both an unsupervised clustering part (k-means algorithm) and a supervised classification part (Support Vector Machine algorithm), working independently. From those two different approaches 83% compatibility is obtained. Such optimization can improve limit of detection (LOD) of glycerol concentrations by a factor of 12 using a QCM with a fundamental resonance frequency of 10 MHz. This approach can be applied not only to biosensors but also to chemical sensors involving resonance frequency monitoring by means of impedance spectroscopy, and has the potential to be expanded to systems with different transducer types, monitoring resonance by other means.

Biosensors for the Detection of Bacillus anthracis.

Bacillus anthracis, present in two forms of vegetative cells and spores, is a pathogen that infects humans through contact with infected animals or contaminated animal products and is also maliciously used in terrorist acts. Therefore, a rapid and sensitive test for B. anthracis is necessary but challenging. The challenge comes from the following aspects: an accurate distinction of B. anthracis from other Bacillus species due to their high genomic similarity and the horizontal gene transfer between Bacillus members; direct detection of the B. anthracis spores without damaging them for component extraction to avoid the risk of spore atomization; and the rapid detections of B. anthracis in complex samples, such as soil and suspicious powders, without sample pretreatments and expensive large-scale equipment. Although culturing B. anthracis from samples is the conventional method for the detection of B. anthracis, it is time-consuming and the detection results would not be easy to interpret because many Bacillus species share similar phenotypic features such as a lack of motility and hemolysis, resistance to gamma phages, and so on. Intensive and extensive effort has been expended to develop reliable detection technologies, among which biosensors exhibit comprehensive advantages in terms of sensitivity, specificity, and portability. Here, we briefly review the research progress, providing highlights of the latest achievements and our own practice and experience. The contents can be summarized in three aspects: the discovery of detection targets, including genes, toxins, and other components; the creation of molecular recognition elements, such as monoclonal antibodies, single-chain antibody fragments, specific peptides, and aptamers; and the design and construction of biosensing systems by the integration of appropriate molecular recognition elements and transducer devices. These sensor devices have their own characteristics and different principles. For example, the surface plasmon resonance biosensor and quartz crystal microbalance biosensor are very sensitive, while the multiplex PCR-on-a-chip can detect multitargets. Biosensors for direct spore detection are highly recommended because they are not only fast but also avoid contamination from aerosol-containing spores. The introduction of nanotechnology has significantly improved the performance of biosensors. Superparamagnetic nanoparticles and phage-displayed gold nanoparticle ligand peptides have made the results of spore detection visible to the naked eye. Because of space constraints, many advanced biosensors for B. anthracis are not described in detail but are cited as references. Although biosensors provide a variety of options for various application scenarios, the challenges have not been fully addressed, which leaves room for the development of more advanced and practical B. anthracis detection means.

Label-Free Quartz Crystal Microbalance Biosensor Based on Aptamer-Capped Gold Nanocages Loaded with Polyamidoamine for Thrombin Detection

Thrombin is an important biomarker, and its detection is of great significance for diagnosis and prevention of related diseases. Conventional methods such as enzyme-linked immunosorbent assay are sensitive but require multiple steps. In this work, a label-free biosensor that only required one step of incubation was developed based on target-triggered release of the cargo molecules from gold nanocages, which achieved highly sensitive and specific detection of thrombin. The proposed biosensor consists of an array of gold nanocages loaded with molecules in their interiors and the DNA probes immobilized on their surface for hybridization with thrombin-specific aptamers to seal their pores. Upon interaction with thrombin, the surface aptamers were lifted off the gold nanocages, resulting in release of the cargo molecules. The loss of cargo molecules was detected by quartz crystal microbalance (QCM). The signal could be amplified by the choice of cargo molecules. The use of polyamidoamine as cargo molecules allowed us to achieve a label-free biosensor with a linear detection range of 0.0086–86 nM and a limit of detection of 7.7 pM. The biosensor was also tested with spiked human serum samples with a limit of detection of 1.2 nM. The detection could be carried out within 1.5 h with only one incubation step of 45 min. The specificity of the biosensor was confirmed by testing against bovine serum albumin and lysozyme at 1 μM.

Disaggregation Behavior of Amyloid β Fibrils by Anthocyanins Studied by Total-Internal-Reflection-Fluorescence Microscopy Coupled with a Wireless Quartz-Crystal Microbalance Biosensor.

Amyloid fibrils are formed from various proteins, some of which cause the corresponding neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. It has been reported that many compounds inhibit the formation of amyloid fibrils. Anthocyanins are flavonoid pigments present in fruits and vegetables, which are known to suppress symptoms related with Alzheimer's disease. However, the influence of anthocyanins on the amyloid fibril remains unclear. Here, we succeeded in the direct monitoring of the disaggregation reaction of single amyloid β (Aβ) fibrils by anthocyanins using total-internal-reflection-fluorescence microscopy with a quartz-crystal microbalance (TIRFM-QCM). It is found that the disassembly activity to the Aβ fibrils depends on the number of hydroxyl groups in six-membered ring B of anthocyanin, and only delphinidin-3-galactoside, possessing three hydroxyl groups there, shows high disassembly activity. Our results show the importance of the number of hydroxyl groups and demonstrate the usefulness of TIRFM-QCM as a powerful tool in studying interactions between amyloid fibrils and compounds.

Sensitive label-free immunoglobulin G detection using a MEMS quartz crystal microbalance biosensor with a 125 MHz wireless quartz resonator

We present a wireless quartz crystal microbalance (QCM) biosensor fabricated using MEMS technology. The MEMS QCM biosensor contains a 125 MHz AT-cut quartz resonator embedded in the microchannel. Because of the compact design, the MEMS QCM biosensor is suitable for mass production and device miniaturization. We performed immunoglobulin G (IgG) detection measurements with different concentrations of IgG. The detection limit was 1 ng ml–1 or less, which is superior to that of the gold-standard surface plasma resonance method. Furthermore, we studied the binding affinity between protein A and IgG by studying the frequency response of the QCM biosensor. We found good agreement with reported values. Therefore, the presented MEMS QCM biosensor has the advantages of compactness, low cost, low power consumption, high sensitivity, and reliability.

Biosensors for Detection of Biochemical Markers Relevant to Osteoarthritis.

This systematic review aimed to assess the advantages of biosensors in detecting biomarkers for the early diagnosis of osteoarthritis (OA). OA is the most prevalent musculoskeletal disease and is a leading cause of disability and pain worldwide. The diagnosis of OA could be performed through clinical examinations and imaging only during the late stages of the disease. Biomarkers could be used for the diagnosis of the disease in the very early stages. Biosensors could detect biomarkers with high accuracy and low costs. This paper focuses on the biosensors mainly adopted to detect OA markers (electrochemical, optical, Quartz crystal microbalance, molecular and wearable biosensors). A comprehensive search on PubMed, Cochrane, CINAHL and Embase databases was conducted from the inception to November 2020. The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines were used to improve the reporting of the review. The Methodological Index for Non-Randomized Studies (MINORS) was used for quality assessment. From a total amount of 1086 studies identified, only 19 articles were eligible for this study. The main advantages of the biosensors reported were accuracy, limited cost and ease of use, compared to traditional methods (ELISA). Otherwise, due to the lack of data and the low level of evidence of the papers included, it was impossible to find significant results. Therefore, further high-quality studies are required.

Homosistein tayini amaçlı QCM-temelli biyosensör

High plasma homocysteine levels can cause an increased risk of cardiovascular, cerebrovascular, and peripheral arterial diseases. Besides this, Alzheimer’s disease and other dementias, osteoporosis, diabetes and renal disease due to folate and B-vitamin deficiency, various drugs or pre-existing atherosclerotic diseases may be the result of high homocysteine levels. The presented research work aimedto perform the detection of Homocysteine (Hcy) by using Quartz Crystal Microbalance (QCM) biosensor. The temperature controlled QCM system was a home-made designed and constructed equipment which can use silver electrod quartz crystal. The modification of silver electrod quartz crystals surfaces was achieved by the surface cleaning process with sodium hydroxide, acetone and methanol in a consecutive manner. Then self-assembled monolayer of cysteamine and chemical coupling of glutaraldehyde (GA) to free end of monolayer was achieved to create the new functional surface in order to complete the formation of spacer arm/ligand. Homocysteine specific recognizing ligand, anti-Homocysteine antibody was immobilized to glutaraldehyde coupled surfaces. The change in resonance frequency values were measured for each modification step. The optimization of dilution ratio of the antibody solution was performed to modified surfaces. The least dilution ratio of antibody, 1/10000 v/v, was determined as optimum antibody ratio. The detection of homocystein was analysed at a detection limit of 0.1 µM and the linear ranges of calibration curves were estimated as 0.1-2.0 µM and 10-50 µM. Homocysteine values indicated good linearities (R2=0.9813 and 0.9875, respectively). The relative standart deviation (RSD %) for precision was calculated as less than 10%. In conclusion, it was found that the detection of homocysteine can be done both in nano- and micro-molar concentration levels. Additionally, designed biosensor showed desired stability and reproducibility. Finally, a new method different from the present methods for the use in the analysis of Hcy was proposed and developed which detects homocysteine by designed QCM technique with a rapid, cheeper and less pretreatment processes. Additionally, homocysteine detection was performed in nano- and micro- molar concentration values.

Determination of plasmin in milk using QCM and ELISA methods

Quartz crystal microbalance (QCM) biosensor for detection plasmin (PLA) in milk using β-casein as a substrate immobilized at piezocrystal surface was reported. The cleavage of β-casein induced by PLA within the concentration range 0.1–40 nM resulted in an increase of series resonant frequency, fs, and a decrease of motional resistance, Rm, due to removal of short peptide fragments from the crystal surface and decrease of viscosity contribution, respectively. The sensor was verified in both raw and spiked milk samples of various origins (cow, goat, sheep). The sensor response has also been studied for trypsin and α-chymotrypsin activities. Sensor technology achieved a limit of PLA detection (LOD) 167.16 ± 39.36 pM at pH 7.4. Similar LOD, 121.98 ± 18.30 pM, was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) test performed in-parallel with the biosensor measurements. Atomic force microscopy (AFM) confirmed cleavage effect of PLA on the surface covered by β-casein.