Quartz Crystal Microbalance Biosensor Research Articles

Determination of E. coli by a Graphene Oxide-Modified Quartz Crystal Microbalance

ABSTRACTA biosensor for the determination of Escherichia coli using graphene oxide on the crystal (gold) surface was fabricated by the drop cast method. The E. coli sensing characteristics of the biosensor, such as a change in frequency, were examined by exposing the graphene oxide-coated crystal to various functionalization steps at room temperature. Graphene oxide was functionalized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride–N-hydroxysuccinimide to covalently conjugate β-galactosidase antibodies to recognize microorganisms that produce this material. Frequency changes in the quartz crystal microbalance are dependent on the absorbed/desorbed masses of the analytes on the functional surface of the crystal. In addition, various characterization techniques were optimized for the morphological elemental analysis of the nanocoating that included field emission scanning electron microscopy, scanning electron microscopy, and electron diffraction spectroscopy. This surface was used in a quartz crystal microbalance nanoplatform for the rapid, sensitive, and label-free detection of E. coli. Under optimal conditions, the frequency of quartz crystal microbalance biosensor was directly proportional to the concentration of antigen with a dynamic range from 0.5 mg mL−1 to 5 ng mL−1 and a minimum detection limit of 5 ng mL−1, and a sensitivity of 0.037 Hz g ml−1 cm−1. These results show that the graphene oxide-coated crystal had excellent performance for E. coli. This research reports a simple, inexpensive, and effective highly stable biosensor using graphene oxide as the sensing medium.

EFFECT OF UV RADIATION DURATION AND MOLECULAR WEIGHT TO HYDROPHOBICITY AND SURFACE ROUGHNESS OF POLYSTYRENE COATING ON QCM SENSOR

Hydrophobicity is one of important solid surface properties for the development of Quartz Crystal Microbalance (QCM) biosensor. Hydrophobicity plays a role in the biomolecule immobilisation. Polystyrene is one of the coating materials used in the QCM biosensor, where the sensitive biomolecule material is immobilised. Hydrophobicity and surface roughness can be controlled by many methods. In this work, we investigated the effect of the polymer molecular weight and UV radiation on the surface roughness and hydrophobicity. The polystyrene with a molecular weight of 35,000 g/mol, 192,000 g/mol, and 280.000 g/mol were solved in toluene with a concentration of 3%, 5%, and 7% and coated using spin coating method on top of the QCM sensor. The coated polystyrene on QCM sensor was irradiated using UV lamp with a wavelength of 254 nm. The contact angle of water before and after UV irradiated was measured using contact angle instrument and the surface roughness is measured using non-contact optical profilometer. The result shows that the higher molecular weight of polystyrene led to more hydrophobic surface. Radiation under UV light increases the hydrophobicity of polystyrene surface. The surface roughness of the polystyrene on top of the sensor is not affected by UV irradiation.

Probing the Dynamic Interaction between Damaged DNA and a Cellular Responsive Protein Using a Piezoelectric Mass Biosensor.

The binding events between damaged DNA and recognition biomolecules are of great interest for understanding the activity of DNA-damaging drugs and the related DNA repair networks. Herein, a simple and sensitive sensor system was tailored for real-time probing of the dynamic molecular recognition between cisplatin-damaged-DNA (cisPt-DNA) and a cellular responsive protein, high-mobility-group box 1 (HMGB1). By integration of flow injection analysis (FIA) with quartz crystal microbalance (QCM), the interaction time-course of cisPt-DNA and HMGB1 domain A (HMGB1a) was investigated. The highly specific sensing interface was carefully designed and fabricated using cisPt-DNA as recognition element. A hybrid self-assembled monolayer consisting of cysteamine and mercaptohexanol was introduced to resist nonspecific adsorption. The calculated kinetic parameters (kass and kdiss) and the dissociation constant (KD) demonstrated the rapid recognition and tight binding of HMGB1a toward cisPt-DNA. Molecular docking was employed to simulate the complex formed by cisPt-DNA and HMGB1a. The tight binding of such a DNA-damage responsive complex is appealing for the downstream molecular recognition event related to the resistance to DNA repair. This continuous-flow QCM biosensor is an ideal tool for studying specific interactions between drug-damaged-DNAs and their recognition proteins in a physiological-relevant environment, and will provide a potential sensor platform for rapid screening and evaluating metal anticancer drugs.

Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.

Monitoring the Cellular Binding Events with Quartz Crystal Microbalance (QCM) Biosensors.

Quartz crystal microbalance (QCM) biosensors have been demonstrated as noninvasive and label-free tools for cell based measurements. However, the complexity of biofilms composed of cells with the associated liquid environments is preventive towards forming explicit relationship between the added mass and the change in the frequency output signal. Therefore, the protocols of interface design (surface chemistry, interaction mechanism, and data acquisition), data interpretation, and device fabrication, all need to be finely refined in order to make these biosensors prevail in real life. Especially in the sense of deriving correct inferences from binding events, the fluidic effects (mostly visible in the form of damping resistance of QCM) should be quantitatively excluded from binding measurements. Such strategies can then track even the cellular interactions which are the basis of many physiological functions of life and can be built into smart functional devices for point of care diagnostics. This chapter provides technical details regarding these strategies with a focus on experimental details and procedures of the measurements of anti CD-20 antibody (Rituximab) interactions with B-Lymphoma cancer cells using the QCM method. In addition to a detailed description of specific interactions, we provide mechanisms of data interpretation and device development having potential application to other techniques.

Magnesium Zinc Oxide Nanostructure-modified Quartz Crystal Microbalance for Dynamic Monitoring of Antibiotic Effects and Antimicrobial Resistance

We demonstrate a magnesium zinc oxide (MZO) nanostructure-modified quartz crystal microbalance (MZOnano-QCM) biosensor to dynamically monitor antimicrobial effects on E. coli and S. cerevisiae. The MZO nanostructures were grown on the top electrode of a standard QCM using metal-organic chemical-vapor deposition (MOCVD). The MZO nanostructures are utilized as the sensing material due to their multifunctionality, biocompatibility, and very large effective sensing surface. The MZO surface-wettability and morphology are controlled, offering high-sensitivity to various biological/biochemical species. The MZOnano-QCM was applied to detect the effects of ampicillin and tetracycline on sensitive and resistant strains of E.coli, as well as effects of amphotericin-B and miconazole on S. cerevisiae through the device's time-dependent frequency shift and motional resistance.

Development and application of DNA molecular probes

The development of DNA probes started from 1950's for diagnostic purposes and it is still growing. DNA probes are applied in several fields such as food, medical, veterinary, environment and security, with the aim of prevention, diagnosis and treatment. The use of DNA probes permits microorganism identification, including pathogen detection, and their quantification when used in specific systems. Various techniques obtained success by the utilization of specific DNA probes, that allowed the obtainment of rapid and specific results. From PCR, qPCR and blotting techniques that were first used in well equipped laboratories to biosensors such as fiber optic, surface plasmon resonance (SPR), electrochemical, and quartz crystal microbalance (QCM) biosensors that use different transduction systems. This review describes i) the design and production of primers and probes, and their utilization from the traditional techniques to the new emerging techniques like biosensors used in current applications; ii) the possibility to use labelled-free probes and probes labelled with an enzyme/fluorophore, etc.; iii) the different sensitivity obtained by using specific systems; and iv) the advantage obtained by using biosensors.

Gold Nanoparticles Assembly on Silicon and Gold Surfaces: Mechanism, Stability, and Efficiency in Diclofenac Biosensing

We investigated the assembly of gold nanoparticles (AuNPs) on gold and silicon sensors with two final objectives: (i) understanding the factors governing the interaction and (ii) building up a nanostructured piezoelectric immunosensor for diclofenac, a small-sized pharmaceutical pollutant. Different surface chemistries were devised to achieve AuNPs assembly on planar substrates. These surface chemistries included amines to immobilize AuNPs via electrostatic interaction or a mixture of amines and thiols to covalently attach the AuNPs. We also generated PEG-amine-terminated surfaces to benefit from the well-known non-biofouling properties of PEG-coated surfaces. The functional substrates and the resulting gold nanoparticle layers were characterized in detail by surface IR, contact angle measurements, and scanning electron microscopy (SEM). The mechanism of adsorption is discussed herein considering the nature of the terminal groups and their charge at the pH of AuNPs adsorption. The coverage and the dispersion of AuNPs were strongly dependent on the anchoring points on the surfaces; the optimal were reached when the attachment layer offered multiple interaction points, in particular, for NH2/SH- and PEG/NH2-terminated surfaces, where the percentage of isolated particles was up to 78%. In addition, PEG-coated surfaces led to a stable AuNPs layer resistant to ultrasounds and to further functionalization of the immobilized nanoparticles. These surfaces were used to engineer quartz crystal microbalance (QCM) biosensors for diclofenac detection. The AuNPs nanostructured substrates significantly enhanced the biosensor sensitivity as compared to planar substrates (up to 6 times higher). This enhancement presages a higher sensitivity in the competitive detection of diclofenac on these systems. More importantly, despite the biorecognition and the drastic regeneration conditions, SEM images show that gold nanoparticles layers are stable and reliable, which paves the way for their use as nanostructured platforms for multiple applications.

Use of Piezo-Immunosensors in Detecting Antigens and Antibodies Using Quartz Crystal Microbalance (QCM): a Novel Technique for Characterization, Understanding Interactions and Mechanisms of Action and Resistance of Monoclonal Antibodies

Background: Antigens like CD20 antigen, are established targets for antibody therapy with monoclonal antibodies (mAb) like Rituximab. Mixed clinico-pathological responses to mAb have been reported either due to presence of antibodies, rapid clearance or low density of target receptor/antigens. This demands need for an assay to monitor serum mAb therapeutic levels to ensure appropriate dosage. Enzyme-linked immunosorbent assay (ELISA) is still the most widely used technique to detect mAb level in human serum which is expensive and time consuming. Understanding properties and interactions of antigens is quintessential for developing better targeted agents and overcoming resistance. Flow cytometry is still the most widely used technique to detect CD20 level in human serum which is expensive, time consuming and does not reveal any details of interaction between the molecules. We have developed a new innovative biosensor based novel technique to not only monitor levels but also study real time interaction of antigens with antibodies using QCM Piezo-immunosensor. This quantitative label free peptide based assay can be used to characterize cell surface antigen, to study antigen- antibody interactions and obtain understanding of mechanisms of resistance to therapy.Method:Mimotope was used as a substitute for the antigen like CD20 and HER2 receptor protein in QCM assays to detect mAb level. The validation samples were prepared from the standard T solution in 10% human serum at three concentrations (10, 20 and 40 ug/ml). The changes in frequencies (ΔF) of sera from 3 female patients were obtained by calculating the differences between frequency shifts in pre and post mAb infusion. mAb level was calculated by equation, (ΔF +1.0022) ÷ 0.9997 μg / ml. The real-time processes of attachment of Cells like Raji cells on the gold electrode and the subsequent binding of antibody like Rituximab to the cells were studied using QCM biosensor. The interaction between Antigen and Antibody led to the increased resonant frequency shifts (df0) in the studied antibody concentration range from 5 to 250 μg mL-1 . Control experiments using other therapeutic antibodies (i.e., Trastuzumab and Bevacizumab) and different cells (i.e., T cells and endothelial cells) proved very specific interaction between Rituximab and CD20 antigens on B cellsResults: Antigen and Antibody binding was very specific. This binding decreased the electrochemical activity and stability of the cells, supporting the cell lysis mechanisms of action of Rituximab. We showed that assay sensitivity was dependent upon the amino acids used to tether and link the peptide to the sensor surface and the buffers used. QCM assay was capable of detecting mAb like Trastuzumab serum level as low as 0.038 nM (linear operating range of 0.038-0.859 nM). The time frame of assay was 20-30 minutes. These results were in concordance with previously published results using ELISA. We have shown a systematic approach for using QCM technique to quantify the apparent binding constant between antigen and antibody can reveal antigen density.Conclusion: We have established a low cost, highly sensitive, fast, synthetic peptide based QCM assay which could be used as a basis for developing a new generation of affinity-based Immunosensor assays to monitor mAb serum levels like Rituximab, Trastuzumab and other monoclonal antibodies, helping physicians to determine the clinical efficacy of these drugs and ensuring appropriate dosages. Moreover antigen density and interactions of antigens with respective monoclonal antibodies like CD20 with Rituximab will help physicians to determine the clinical efficacy and resistance mechanisms to targeted antibodies like Rituximab and Ofatumumab. This could be used as a basis for developing a new generation of affinity-based Immunosensor assays. Our study shows that peptide mimotopes have potential benefit in sensor applications as the peptide-peptide interactions in the peptide mimotopes could be manipulated by the addition of functional groups to the peptide to influence binding of the target protein as well as for surface immobilization. DisclosuresNo relevant conflicts of interest to declare.

Dynamic monitoring of antimicrobial resistance using magnesium zinc oxide nanostructure-modified quartz crystal microbalance

Antimicrobial resistance (AMR) is becoming a major global-health concern prompting an urgent need for highly-sensitive and rapid diagnostic technology. Traditional assays available for monitoring bacterial cultures are time-consuming and labor-intensive. We present a magnesium zinc oxide (MZO) nanostructure-modified quartz crystal microbalance (MZOnano-QCM) biosensor to dynamically monitor antimicrobial effects on E. coli and S. cerevisiae. MZO nanostructures were grown on the top electrode of a standard QCM using metal-organic chemical-vapor deposition (MOCVD). The MZO nanostructures are chosen for their multifunctionality, biocompatibility, and giant effective sensing surface. The MZO surface-wettability and morphology are controlled, offering high-sensitivity to various biological/biochemical species. MZO-nanostructures showed over 4-times greater cell viability over ZnO due to MZO releasing 4-times lower Zn2+ density in the cell medium than ZnO. The MZOnano-QCM was applied to detect the effects of ampicillin and tetracycline on sensitive and resistant strains of E.coli, as well as effects of amphotericin-B and miconazole on S. cerevisiae through the device's time-dependent frequency shift and motional resistance. The MZOnano-QCM showed 4-times more sensitivity over ZnOnano-QCM and over 10-times better than regular QCM. For comparison, the optical density at 600nm (OD600) method and the cell viability assay were employed as standard references to verify the detection results from MZOnano-QCM. In the case of S. cerevisiae, the OD600 method failed to distinguish between cytotoxic and cytostatic drug effects whereas the MZOnano-QCM was able to accurately detect the drug effects. The MZOnano-QCM biosensor provides a promising technology enabling dynamic and rapid diagnostics for antimicrobial drug development and AMR detection.

Multifunctional oligomer incorporation: a potent strategy to enhance the transfection activity of poly(l-lysine).

Natural polycations, such as poly(l-lysine) (PLL) and chitosan (CS), have inherent superiority as non-viral vectors due to their unparalleled biocompatibility and biodegradability. However, the application was constrained by poor transfection efficiency and safety concerns. Since previous modification strategies greatly weakened the inherent advantages of natural polycations, developing a strategy for functional group introduction with broad applicability to enhance the transfection efficiency of natural polycations without compromising their cationic properties is imperative. Herein, two uncharged functional diblock oligomers P(DMAEL-b-NIPAM) and P(DMAEL-b-Vlm) were prepared from a lactose derivative, N-iso-propyl acrylamide (NIPAM) as well as 1-vinylimidazole (Vlm) and further functionalized with four small ligands folate, glutathione, cysteine and arginine, respectively, aiming to enhance the interactions of complexes with cells, which were quantified utilizing a quartz crystal microbalance (QCM) biosensor, circumventing the tedious material screening process of cell transfection. Upon incorporation with PLL and DNA, the multifunctional oligomers endow the formulated ternary complexes with great properties suitable for transfection, such as anti-aggregation in serum, destabilized endosome membrane, numerous functional sites for promoted endocytosis and therefore robust transfection activity. Furthermore, different from the conventional strategy of decreasing cytotoxicity by reducing the charge density, the multifunctional oligomer incorporation strategy maintains the highly positive charge density, which is essential for efficient cellular uptake. This system develops a new platform to modify natural polycations towards clinical gene therapy.

Silver nanocluster based sensitivity amplification of a quartz crystal microbalance gene sensor

The article describes a quartz crystal microbalance (QCM) biosensor for the determination of nucleic acids via a DNA-templated assembly of silver nanoclusters (AgNCs) which represents a novel way for efficient signal amplification. A QCM was modified with probe DNA to specifically capture target DNA. Then, DNA-templated AgNCs were assembled to enhance the sensitivity of the QCM sensor via Ag(I) ions attached to the DNA skeleton, this followed by hydroquinone-induced reductive formation of the AgNCs. TEM and AFM were used to further confirm the formation of DNA-templated AgNCs. The results showed that frequency response of QCM sensor is up to 87 times larger when using this mode of amplification. A linear relationship was obtained between the frequency response and DNA concentration over the 0.6 to 130 nM range, with a 0.1 nM detection limit. In our perception, this scheme for improved sensitivity provides a straightforward and widely applicable tool for sensing DNA.

Identification of a peptide ligand for antibody immobilization on biosensor surfaces

The construction of biosensors with antibodies as the biological sensing element requires the integration of antibodies into the biosensor such that their activity is reproducibly retained. In this work, a peptide ligand was identified from phage-displayed heptameric peptide libraries for the purpose of antibody immobilization on biosensor surfaces. For biopanning of peptides specific to the Fc region of the antibody, rabbit anti-goat immunoglobulin G (IgG) antibody was immobilized on a culture dish coated with goat IgG, which enabled exposure of the Fc region of rabbit antibody on the surface with the Fab region bound to the surface-adsorbed goat IgG. The library phages were added to the dish and phages displaying antibody-binding peptides were competitively eluted using the rabbit antibody. After four rounds of biopanning, 22 phage plaques were randomly selected and their nucleotide sequences corresponding to the random peptide were determined. Three enriched sequences were selected and analyzed for their rabbit antibody-binding activity. One of these sequences (KHRFNKD), which was rich in positive charge and homologous to the IgG-binding domains of Staphylococcal protein A, exhibited a high and specific affinity to rabbit antibody. This peptide was immobilized on a quartz crystal microbalance (QCM) biosensor, and the resulting QCM was found to be capable of capturing antibody and detecting antigen, with potential applications in antibody purification and enzyme immunoassay.

The evaluation of loop-mediated isothermal amplification-quartz crystal microbalance (LAMP-QCM) biosensor as a real-time measurement of HPV16 DNA

We have previously developed quartz crystal microbalance biosensor integrated with loop-mediated isothermal amplification (LAMP-QCM) for human papillomavirus (HPV) type58 DNA detection. Infection with HPV, particularly HPV16, remains a serious health problem due to its major risk factor contributing to cervical cancer. In the present study, LAMP-QCM biosensor was evaluated in terms of a quantitative assay for copy number of HPV16 DNA in cervical samples compared to quantitative PCR using TaqMan assay (TaqMan-qPCR). The detection limit of LAMP-QCM was found to be 10 fold more sensitive than TaqMan-qPCR with 100% specificity and 7.6% imprecision. Different plot of HPV16 DNA copy number using Bland–Altman analysis revealed 94% correlation between LAMP-QCM and qPCR. We therefore concluded that the developed LAMP-QCM biosensor provides a possible rapid and sensitive assay for HPV16 DNA quantification in a routine laboratory.

Oriented and reversible immobilization of His-tagged proteins on two- and three-dimensional surfaces for study of protein–protein interactions by a QCM biosensor

A two-dimensional (2D) and a three-dimensional (3D) His-tag capture surfaces were fabricated for oriented and reversible immobilization of His-tagged proteins on quartz crystal microbalance (QCM) biosensor surfaces, which can be used for label-free and real-time detection of the interactions between His-tagged protein and its interacting protein (analyte). His-tagged proteins immobilized on the 2D His-tag capture surface maintained a higher binding activity than those immobilized on a 2D carboxyl surface via amine coupling. The 3D His-tag capture surface has about twice the amount of immobilization capacity as the 2D His-tag capture surface, which enables a higher sensitivity for detection. His-tag capture surface can be optionally regenerated to remove the His-tagged protein as well as the analyte for the next cycle of His-tagged protein immobilization, or to only selectively remove the analyte, leaving the His-tagged protein on the surface for the next cycle of analyte binding. Furthermore, the kinetic and affinity studies of the interactions between the His-tagged protein and its interacting protein were performed. This study provides an efficient way to study protein–protein interactions by oriented and reversible immobilization of His-tagged proteins on QCM biosensor surfaces.

Development of effective QCM biosensors by cyclopropylamine plasma polymerization and antibody immobilization using cross-linking reactions

Biosensors have been extensively developed and applied for biomedical and environmental studies. Although there are many different types of biosensing techniques, immobilization of the biorecognition biomolecules onto the sensor surface is always required. Cyclopropylamine pulsed plasma polymerization in radio frequency (RF) capacitive discharges was employed to deposit amine thin films on the gold electrode of quartz crystal microbalance (QCM) biosensors and the monoclonal antibody AL-01, specific to human serum albumin (HSA), was immobilized onto the plasma polymer surface. Two different amine plasma polymers were studied. They had a similar amount of primary amine groups, 1.3–1.5 at.%, as determined by chemical derivatization but their stability in water was different. The 1st type, deposited at the floating potential and 120 Pa, exhibited 16% thickness loss after 24 h in water whereas the 2nd type deposited at RF electrode and 50 Pa was completely stable (2% thickness loss). Glutaraldehyde (GA) coupling of AL-01 was employed for both types of the amine films and the performance of QCM immunosensors was evaluated by the immunoassay flow test. A high-stability of the frequency signal was obtained in both the cases but the 2nd type of the film provided low response to HSA. It was explained by its highly cross-linked structure and steric hindrance of active sites. Three different antibody immobilization methods were explored for the 1st type of the film. Stable baseline, selective and high response were recorded for GA and sulfo-SMCC methods. The results confirmed that the presented methodologies for the grafting of biomolecules on the gold are highly efficient and very promising for future use in biosensing.

Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor.

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery.

Viscoelasticity evolution in protein layers during binding reactions evaluated using high-frequency wireless and electrodeless quartz crystal microbalance biosensor without dissipation

In this study, we demonstrate the effectiveness of a resonance acoustic microbalance with a naked embedded quartz (RAMNE-Q) biosensor for evaluating viscoelastic property changes in thin protein layers during protein deposition reactions without dissipation measurement. Quartz crystal microbalance (QCM) biosensors have conventionally been adopted for the viscoelasticity evaluation of adsorbed protein layers by measuring dissipation as well as resonance frequency. However, dissipation, or the vibrational energy loss, is easily affected by many factors and is rarely measured with sufficiently high accuracy. To evaluate viscoelasticity only from a reliable frequency response, one needs to perform an ultrahigh-frequency measurement, which is here achieved using the RAMNE-Q biosensor. Simultaneous frequency measurement is performed for fundamental and overtone modes up to 406 MHz of a 58 MHz RAMNE-Q biosensor during various binding reactions, and evolutions of viscosity, shear modulus, and thickness of adsorbed protein layers are inversely evaluated. A marked difference is observed in the viscosity evolution between specific and nonspecific binding reactions. Furthermore, the reversed frequency response appears, which indicates the modification of the protein structure into a rigid structure.

Nucleation–fibrillation dynamics of Aβ1-40 peptides on liquid–solid surface studied by total-internal-reflection fluorescence microscopy coupled with quartz-crystal microbalance biosensor

We have successfully developed the total-internal-reflection-fluorescence microscopy combined with a quartz-crystal microbalance (TIRFM-QCM) biosensor, and monitored the nucleation–fibrillation phenomenon of amyloid β1-40 peptide on the naked quartz surface. The cross-β-sheet structures were visualized with the TIRFM using the thioflavin-T (Th-T) label, and other unlabeled aggregates were detected through the frequency change of the 58-MHz wireless-electrodeless QCM throughout the aggregation reaction. The QCM response indicates significant adsorption of the peptides on the quartz surface at the early stage, which is followed by fibrillation. The non-cross-β-sheet oligomers are first formed, and nuclei appear in the oligomer region, from which fibrils originate and elongate. The two-color TIRFM observation was performed after the aggregation reaction with the Nile-red label as well as the ThT label for identifying nucleation from non-β-sheet regions. An aggregation model is proposed.

A novel assay for detecting canine parvovirus using a quartz crystal microbalance biosensor

Rapid and accurate diagnosis is crucial to reduce both the shedding and clinical signs of canine parvovirus (CPV). The quartz crystal microbalance (QCM) is a new tool for measuring frequency changes associated with antigen–antibody interactions. In this study, the QCM biosensor and ProLinker™ B were used to rapidly diagnosis CPV infection. ProLinker™ B enables antibodies to be attached to a gold-coated quartz surface in a regular pattern and in the correct orientation for antigen binding. Receiver operating characteristics (ROC) curves were used to set a cut-off value using reference CPVs (two groups: one CPV-positive and one CPV-negative). The ROC curves overlapped and the point of intersection was used as the cut-off value. A QCM biosensor with a cut-off value of −205Hz showed 95.4% (104/109) sensitivity and 98.0% (149/152) specificity when used to test 261 field fecal samples compared to PCR. In conclusion, the QCM biosensor described herein is eminently suitable for the rapid diagnosis of CPV infection with high sensitivity and specificity. Therefore, it is a promising analytical tool that will be useful for clinical diagnosis, which requires rapid and reliable analyses.