A quartz crystal microbalance based study reveals living cell loading rate via αvß3 integrins.

Cellular interactions with the microenvironment are mediated by ligand-receptor bonds. Such ligand-receptor bond dynamics is known to be heavily dependent on the loading rate. However, the physiologically-relevant loading rate of living cells remains unknown. Here, using a quartz crystal microbalance (QCM), we developed a bulk-force sensing platform to semi-quantitatively detect the rate of cellular force application during early stages of cell adhesion and spreading. Atop an Au-coated quartz crystal, covalently linked self-assembled monolayers (SAM) were used to immobilize cyclic-RGDfK peptides that can interact with the αvß3 integrins on cells. The QCM detects the changes in resonant frequency of the vibrating crystal due to the cellular activity/probing (force application) on the QCM surface. The corresponding changes in mass on the surface, proportional to the rate of force application, arise from the cellular interactions with the functionalized surface were calculated. The loading rate of living cells was found to be ∼80-115 pN/s. Collectively, our results revealed a fundamental feature of cell adhesion and spreading providing valuable information regarding cellular interactions with the extracellular matrix.

Design of engineered surfaces for prospective detection of SARS-CoV-2 using quartz crystal microbalance-based techniques.

INTRODUCTION: Rapid transmission of the severe acute respiratory syndrome coronavirus 2 has affected the whole world and forced it to a halt (lockdown). A fast and label-free detection method for the novel coronavirus needs to be developed along with the existing enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR)-based methods. AREAS COVERED: In this report, biophysical aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein are outlined based on its recent reported electron microscopy structure. Protein binding sites are analyzed theoretically, which consisted of hydrophobic and positive charged amino acid residues. Different strategies to form mixed self-assembled monolayers (SAMs) of hydrophobic (CH3) and negatively charged (COOH) groups are discussed to be used for the specific and strong interactions with spike protein. Bio-interfacial interactions between the spike protein and device (sensor) surface and its implications toward designing suitable engineered surfaces are summarized. EXPERT OPINION: Implementation of the engineered surfaces in quartz crystal microbalance (QCM)-based detection techniques for the diagnosis of the novel coronavirus from oral swab samples is highlighted. The proposed strategy can be explored for the label-free and real-time detection with sensitivity up to ng level. These engineered surfaces can be reused after desorption.

In-line detection of monoclonal antibodies in the effluent of protein A chromatography with QCM sensor.

A major drawback of the IgG capture step is the high cost of the protein A resin. For a better utilization of the resin, a continuous multi-column operation was recently proposed. In this method, accurate detection of leaking IgG is crucial to divert the breakthrough fluid from the waste to the next column and prolong the loading step without product loss. The detection of a breakthrough point as a change in UV absorption is based on a relatively small signal addition of IgGs to the bulk signal of host cell proteins. To achieve specificity, we used a quartz crystal microbalance and immobilized protein A as specific ligand on the sensor surface. We integrated the quartz crystal microbalance sensor in-line after the protein A column for real-time detection of IgGs in the breakthrough fluid. We show that this specific IgG detection in the breakthrough fluid can be more sensitive than with the UV detector. The use of the same product-specific ligand in the affinity column and in the sensor allows simultaneous in-line regeneration of column and sensor in a single step. Such a sensor could support cost-efficient load control during the entire continuous multi-column capture step in downstream processing.

Application of QCM in Peptide and Protein-Based Drug Product Development.

AT-cut quartz crystals vibrating in the thickness-shear mode (TSM), especially quartz crystal resonators (QCRs), are well known as very efficient mass sensitive systems because of their sensitivity, accuracy, and biofunctionalization capacity. They are highly reliable in the measurement of the mass of deposited samples, in both gas and liquid matrices. Moreover, they offer real-time monitoring, as well as relatively low production and operation costs. These features make mass sensitive systems applicable in a wide range of different applications, including studies on protein and peptide primary packaging, formulation, and drug product manufacturing process development. This review summarizes the information on some particular implementations of quartz crystal microbalance (QCM) instruments in protein and peptide drug product development as well as their future prospects.

Real-Time Monitoring of the Regulatory Volume Decrease of Cancer Cells: A Model for the Evaluation of Cell Migration.

Cell migration plays a vital role in carcinoma invasion and metastasis. Cell regulatory volume decrease (RVD), a mechanism of adjusting cell volume, is a basic physiological function of cells, which is closely related to cell migration. In this work, a quartz crystal microbalance (QCM) cytosensor was first developed for real-time monitoring of cell RVD to evaluate the migration of human breast cancer cells. While stimulating the immobilized cells on the chip with hypotonic solutions, the temporal dynamics of RVD can be tracked by QCM sensor via analyzing frequency shifts during the cell swelling and shrinkage. The results showed that, due to the difference in cell migration capability, the level of RVD for MCF-7 cells and MDA-MB-231 cells was 32.8 ± 2.9% and 49.7 ± 4.2% ( n = 3), respectively. Furthermore, tamoxifen, a chloride channel blocker, was used to suppress cell RVD, indicating concentration dependence and inhibition difference in both types of cells. Combining QCM measurement with cell migration assay, the results showed that the blockage of RVD was positively correlated to the inhibition of cell migration with tamoxifen concentration ranging from 5 to 60 µM, which revealed the relation between cell RVD and cell migration. The study provided a noninvasive and real-time strategy for monitoring cell RVD as well as assessing cell migration, which was expected to supply a new diagnostic tool for metastatic cancers.

A molecularly imprinted nanofilm-based quartz crystal microbalance sensor for the real-time detection of pirimicarb.

This study aimed to prepare a novel quartz crystal microbalance (QCM) sensor for the detection of pirimicarb. Pirimicarb-imprinted poly (ethylene glycol dimethacrylate-N-metacryloyl-(l)-tryptophan methyl ester) [p (EGDMA-MATrp)] nanofilm (MIP) on the gold surface of a QCM chip was synthesized using the molecular imprinting technique. A nonimprinted p (EGDMA-MATrp) nanofilm (NIP) was also synthesized using the same experimental technique. The MIP and NIP nanofilms were characterized via Fourier transform infrared spectroscopy attenuated total reflectance spectroscopy, contact angle, atomic force microscopy, and an ellipsometer. A competitive adsorption experiment on the sensor was performed to display the selectivity of the nanofilm. An analysis of the QCM sensor showed that the MIP nanofilm exhibited high sensitivity and selectivity for pirimicarb determination. A liquid chromatography-tandem mass spectrometry method was prepared and validated to determine the accuracy and precision of the QCM sensor. The accuracy and precision of both methods were determined by a comparison of six replicates at three different concentrations to tomato samples extracted by using a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method. The limit of detection of the QCM sensor was found to be 0.028 nM. In conclusion, the QCM sensor showed good accuracy, with recovery percentages between 91 and 94%. Also, the pirimicarb-imprinted QCM sensor exhibited a fast response time, reusability, high selectivity and sensitivity, and a low limit of detection. Therefore, it offers a serious alternative to the traditional analytical methods for pesticide detection in both natural sources and aqueous solutions.

Preparation of a molecularly imprinted sensor based on quartz crystal microbalance for specific recognition of sialic acid in human urine.

A novel molecularly imprinted quartz crystal microbalance (QCM) sensor was successfully prepared for selective determination of sialic acid (SA) in human urine samples. To obtain the QCM sensor, we first modified the gold surface of the QCM chip by self-assembling of allylmercaptane to introduce polymerizable double bonds on the chip surface. Then, SA molecularly imprinted polymer (MIP) nanofilm was attached to the modified QCM chip surface. For comparison, we have also characterized the nonmodified and improved surfaces of the QCM sensor by using atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy. We then tested the selectivity and detection limit of the imprinted QCM sensor via a series of adsorption experiments. The results show a linear response in the range of 0.025-0.50 µmol L-1 for sialic acid. Moreover, the limit of detection (LOD) of the prepared imprinted QCM sensor was found to be 1.0 nmol L-1 for sialic acid, and high recovery values range from 87.6 to 108.5% with RSD < 8.7 (n = 5) for the spiked urine sample obtained. Overall, this work presents how a novel QCM sensor was developed and used to detect sialic acid in human urine samples. Graphical abstract Specific recognition of sialic acid by the MIP-QCM sensor system.

Advances in microRNA analysis.

MicroRNAs (miRNAs) are single-stranded noncoding RNA molecules that act as key regulators of mRNA expression and are emerging biomarkers for disease. Their small size (18-25 nt) presents challenges to molecular recognition, labeling, and signal generation. Recent research activity in this field has aimed at the development of methods for miRNA quantification that combine high detectability, broad dynamic range, practicality, multiplexity, and low cost for prospective applications in diagnostic laboratories. This review article covers the most recent advances in microRNA analysis.

A high-throughput QCM chip configuration for the study of living cells and cell-drug interactions.

In this study, we present a novel design of interference-free, negligible installation-induced stress, suitable for the fabrication of high-throughput quartz crystal microbalance (HQCM) chips. This novel HQCM chip configuration was fabricated using eight independent yet same-batch quartz crystal resonators within a common glass substrate with eight through-holes of diameter slightly larger than that of the quartz resonator. Each quartz resonator's rim was adhered to the inner part of the through-hole via silicone glue to form the rigid (quartz)-soft (silicone)-rigid (glass) structure (RSRS) which effectively eliminates the acoustic couplings among different resonators and largely alleviates the installation-induced stresses. The consistence of the eight resonators was verified by very similar equivalent circuit parameters and very close response slopes to liquid density and viscosity. The HQCM chip was then employed for real-time and continuous monitoring of H9C2 cardiomyoblast adhesions and viscoelastic changes induced by the treatments of two types of drugs: drugs that affect the cytoskeletons, including nocodazole, paclitaxel, and Y-27632, and drugs that affect the contractile properties of the cells: verapamil and different dosages of isoprenaline. Meanwhile, we compared the cytoskeleton affecting drug-induced viscoelastic changes of H9C2 with those of human umbilical vein endothelial cells (HUVECs). The results described here provide the first solution to fabricate HQCM chips that are free from the limitation of resonator number, installation-induced stress, and acoustic interferences among resonators, which should find wide applications in areas of cell phenotype assay, cytotoxicity test, drug evaluation and screening, etc. Graphical abstract Schematic illustration of the principle and configuration of interference-free high-throughput QCM chip to evaluate and screen drugs based on cell viscoelasticity.

Design and Validation of a 150 MHz HFFQCM Sensor for Bio-Sensing Applications.

Acoustic wave resonators have become suitable devices for a broad range of sensing applications due to their sensitivity, low cost, and integration capability, which are all factors that meet the requirements for the resonators to be used as sensing elements for portable point of care (PoC) platforms. In this work, the design, characterization, and validation of a 150 MHz high fundamental frequency quartz crystal microbalance (HFF-QCM) sensor for bio-sensing applications are introduced. Finite element method (FEM) simulations of the proposed design are in good agreement with the electrical characterization of the manufactured resonators. The sensor is also validated for bio-sensing applications. For this purpose, a specific sensor cell was designed and manufactured that addresses the critical requirements associated with this type of sensor and application. Due to the small sensing area and the sensor's fragility, these requirements include a low-volume flow chamber in the nanoliter range, and a system approach that provides the appropriate pressure control for assuring liquid confinement while maintaining the integrity of the sensor with a good base line stability and easy sensor replacement. The sensor characteristics make it suitable for consideration as the elemental part of a sensor matrix in a multichannel platform for point of care applications.

Integration of Quartz Crystal Microbalance-Dissipation and Reflection-Mode Localized Surface Plasmon Resonance Sensors for Biomacromolecular Interaction Analysis.

The combination of label-free, surface-sensitive measurement techniques based on different physical principles enables detailed characterization of biomacromolecular interactions at solid-liquid interfaces. To date, most combined measurement systems have involved experimental techniques with similar probing volumes, whereas the potential of utilizing techniques with different surface sensitivities remains largely unexplored, especially for data interpretation. Herein, we report a combined measurement approach that integrates a conventional quartz crystal microbalance-dissipation (QCM-D) setup with a reflection-mode localized surface plasmon (LSPR) sensor. Using this platform, we investigate vesicle adsorption on a titanium oxide-coated sensing substrate along with the amphipathic, α-helical (AH) peptide-induced structural transformation of surface-adsorbed lipid vesicles into a supported lipid bilayer (SLB) as a model biomacromolecular interaction. While the QCM-D and LSPR signals both detected mass uptake arising from vesicle adsorption, tracking the AH peptide-induced structural transformation revealed more complex measurement responses based on the different surface sensitivities of the two techniques. In particular, the LSPR signal recorded an increase in optical mass near the sensor surface which indicated SLB formation, whereas the QCM-D signals detected a significant loss in net acoustic mass due to excess lipid and coupled solvent leaving the probing volume. Importantly, these measurement capabilities allowed us to temporally distinguish the process of SLB formation at the sensor surface from the overall structural transformation process. Looking forward, these label-free measurement capabilities to simultaneously probe adsorbates at multiple length scales will provide new insights into complex biomacromolecular interactions.

On the Hydrodynamic Nature of DNA Acoustic Sensing.

In this work we provide strong experimental evidence for the hydrodynamic nature of the acoustic wave/biomolecule interaction at a solid/liquid interface. By using a wide range of DNAs of various sizes and by assuming DNA attachment as discrete particles through a neutravidin/biotin link, we prove experimentally that the acoustic ratio (dissipation/frequency) is directly related to the molecules' intrinsic viscosity [η]. The relationship of [η] to the size and shape of biomolecules is described in general and more specifically for linear dsDNA; equations are derived linking the measured acoustic ratio to the number of dsDNA base pairs for two acoustic sensors, the QCM and Love-wave devices operating at a frequency of 35 and 155 MHz, respectively. Single-stranded DNAs were also tested and shown to fit well to the equation derived for the double-stranded molecules while new insight is provided on their conformation on a surface. Other types of DNA are also shown to fit the proposed model. The current work establishes a new way of viewing acoustic sensor data and lays down the groundwork for a surface technique where quantitative information can be obtained at the nanometer scale regarding the shape and size, i.e., conformation of biomolecules at an interface.

Real-time monitoring of oxidative injury of vascular endothelial cells and protective effect of quercetin using quartz crystal microbalance.

The adhesion, spreading, and proliferation of human umbilical vein endothelial cell line (HUVEC-C) cells, on a gold electrode were monitored using quartz crystal microbalance (QCM) measurements. The viscodensity effect caused by the normal action of the cells led to a decrease of the resonant frequency and increase of the motional resistance. The oxidative injury of HUVEC-C cells appeared immediately with the addition of H2O2, exhibiting the decline of cellular spreading area and cell coverage on the electrode surface and resulting in inverted QCM responses. The injured extent of the cells was found to be related to the content of H2O2. It is found that 0.05 mM quercetin added beforehand in the growth medium could remove completely the oxidative action of 1.0 mM H2O2. Quercetin with increased dosage still exerted a partial protective effect on HUVEC-C cells against oxidative injury induced by 2.5 mM H2O2. The microscope observations, electrochemical measurements, and MTT analysis validate the QCM assay results, indicating that quercetin is a valuable flavonoid anti-oxidant in the precaution and treatment for the oxidative injury of vascular endothelium. Graphical Abstract Upper part: Microscope images (×400) of 7.5×104 HUVEC-C cells adhered to the substrate at 48 h in the presence of H2O2. Middle part: Real-time Δf 0 and ΔR 1 responses to the addition of 7.5×104 HUVEC-C cells onto QCM gold electrode in the presence of H2O2 added at 24 h after the introduction of the cells. Lower part: Microscope images (×400) of 7.5×104 HUVEC-C cells adhered to the substrate at 48 h in the presence of quercetin added at 18 h and H2O2 added at 24 h after the introduction of the cells.

Rational Design of QCM-D Virtual Sensor Arrays Based on Film Thickness, Viscoelasticity, and Harmonics for Vapor Discrimination.

Herein, we demonstrate an alternative strategy for creating QCM-based sensor arrays by use of a single sensor to provide multiple responses per analyte. The sensor, which simulates a virtual sensor array (VSA), was developed by depositing a thin film of ionic liquid, either 1-octyl-3-methylimidazolium bromide ([OMIm][Br]) or 1-octyl-3-methylimidazolium thiocyanate ([OMIm][SCN]), onto the surface of a QCM-D transducer. The sensor was exposed to 18 different organic vapors (alcohols, hydrocarbons, chlorohydrocarbons, nitriles) belonging to the same or different homologous series. The resulting frequency shifts (Δf) were measured at multiple harmonics and evaluated using principal component analysis (PCA) and discriminant analysis (DA) which revealed that analytes can be classified with extremely high accuracy. In almost all cases, the accuracy for identification of a member of the same class, that is, intraclass discrimination, was 100% as determined by use of quadratic discriminant analysis (QDA). Impressively, some VSAs allowed classification of all 18 analytes tested with nearly 100% accuracy. Such results underscore the importance of utilizing lesser exploited properties that influence signal transduction. Overall, these results demonstrate excellent potential of the virtual sensor array strategy for detection and discrimination of vapor phase analytes utilizing the QCM. To the best of our knowledge, this is the first report on QCM VSAs, as well as an experimental sensor array, that is based primarily on viscoelasticity, film thickness, and harmonics.

Analyte induced water adsorbability in gas phase biosensors: the influence of ethinylestradiol on the water binding protein capacity.

An ultra-sensitive gas phase biosensor/tracer/bio-sniffer is an emerging technology platform designed to provide real-time information on air-borne analytes, or those in liquids, through classical headspace analysis. The desired bio-sniffer measures gaseous 17α- ethinylestradiol (ETED) as frequency changes on a quartz crystal microbalance (QCM), which is a result of the interactions of liquid sample components in the headspace (ETED and water) with a biorecognition layer. The latter was constructed by immobilization of polyclonal antiserum against a phenolic A-ring of estrogenic receptors through protein A. The QCM response exhibited stretched exponential kinetics of negative frequency shifts with reversible and "irreversible" components of mass uptake onto the sensor surface in static headspace conditions when exposed to water solutions of ETED over the sensor working range, from 10(-10) to 10(-17) g L(-1). It was shown that the variations in the QCM response characteristics are due to the change of the water-binding capacity of the sensing layer induced by protein transformations initiated by the binding of ETED molecules. This result is well correlated with the natural physiological function of estrogens in controlling the homeostasis of body fluids in living beings.

A gas-phase amplified quartz crystal microbalance immunosensor based on catalase modified immunoparticles.

A novel signal amplification strategy for quartz crystal microbalance (QCM) based on catalytic gas generation was developed to construct an ultrasensitive immunosensor for the detection of proteins (immunoglobulin G, IgG, used as a model). A catalase modified immunoparticle was prepared to form a sandwich-type immunocomplex with the IgG and anti-IgG antibodies that were immobilized on the QCM sensor. The amount of immunoparticles on the sensor surface was thus controlled by the IgG concentration. Then H2O2 was added and catalyzed by catalase for oxygen generation. The generated oxygen replaced some of the liquid on the sensor surface, leading to the change in the shear modulus of the immunocomplex layer and the apparent viscosity and density of the liquid layer. Due to the ultrasensitive response of QCM to these changes, a significant frequency shift related to the IgG concentration was achieved. Different parameters, including the flow cell structure, operation temperature, immunoparticle concentration, and H2O2 concentration were optimized to achieve steady and efficient frequency shifts. Under the optimal conditions, the proposed gas-phase amplified QCM sensor could achieve up to 72 times improvement of detection sensitivity compared to the label-free sensor as a control, in the concentration range of 0.1-3.0 µg mL(-1). The detection limit was also reduced from 236 ng mL(-1) to 51.0 ng mL(-1) at the 3Sblank level.

A Nano-Thin Film-Based Prototype QCM Sensor Array for Monitoring Human Breath and Respiratory Patterns.

Quartz crystal microbalance (QCM) sensor array was developed for multi-purpose human respiration assessment. The sensor system was designed to provide feedback for human respiration. Thorough optimization of measurement conditions: air flow, temperature in the QCM chamber, frequency measurement rate, and electrode position regarding to the gas flow-was performed. As shown, acquisition of respiratory parameters (rate and respiratory pattern) could be achieved even with a single electrode used in the system. The prototype system contains eight available QCM channels that can be potentially used for selective responses to certain breath chemicals. At present, the prototype machine is ready for the assessment of respiratory functions in larger populations in order to gain statistical validation. To the best of our knowledge, the developed prototype is the only respiratory assessment system based on surface modified QCM sensors.

Integration of a miniature quartz crystal microbalance with a microfluidic chip for amyloid beta-Aß42 quantitation.

A miniature quartz crystal microbalance (mQCM) was integrated with a polydimethylsiloxane (PDMS) microfluidic device for on-chip determination of amyloid polypeptide-Aß42. The integration techniques included photolithography and plasma coupling. Aß42 antibody was immobilized on the mQCM surface using a cross-linker method, and the resonance frequency of mQCM shifted negatively due to antibody-antigen binding. A linear range from 0.1 µM to 3.2 µM was achieved. By using matrix elimination buffer, i.e., matrix phosphate buffer containing 500 µg/mL dextran and 0.5% Tween 20, Aß42 could be successfully detected in the presence of 75% human serum. Additionally, high temperature treatments at 150 °C provided a valid method to recover mQCM, and PDMS-mQCM microfluidic device could be reused to some extent. Since the detectable Aß42 concentration could be as low as 0.1 µM, which is close to cut-off value for Alzheimer patients, the PDMS-mQCM device could be applied in early Alzheimer's disease diagnosis.

Quartz crystal microbalance biosensors: prospects for point-of-care diagnostics.

BACKGROUND: A QCM is a label-free and extremely mass-sensitive device, which allows the detection of the binding event between trace medical analytes and bio-receptors on its surface. QCM, the most promising type of biosensors, has attracted much interest due to the inherent benefits over other transducers, including better sensitivity, ease-of-use, integration with compact analytical devices, and economy, and also involving relatively simple technology in its production. Thus, they have great potential with regard to point-of-care (POC) testing for early detection of diseases. MATERIAL AND METHOD: Retrievable articles that related to acoustic type sensing of Pubmed and Science direct database were included. Additionally, abstracts presented at Biosensor World Congress held between 2008 and 2012 were searching to identify relevant clinical trials. RESULTS: All studies demonstrated the opportunity in the use of QCM as a novel diagnostic method. Several attempts have been made to construct integrated systems that show promising application for POC tests. CONCLUSION: This review represents another step to meet challenges, especially in the improved minimization and sensitivity of biosensors. As this work continues, new bioreceptor and biomarkers emerging from the could make it an ideal candidate for cheap POC diagnostic.

Simultaneous biosensing with quartz crystal microbalance with a dissipation coupled-gate semiconductor device.

In this study, we proposed and demonstrated a novel simultaneous analysis system of biosensing by combining a semiconductor-based field effect transistor (FET) with quartz crystal microbalance with a dissipation (QCM-D) monitoring system. Using the combined system, the changes of not only mass and viscoelasticity but also electrical charge for interaction of charged dextran molecules with substrate, recognition of glucose with low molecular weight, and programmed cell death, apoptosis, were simultaneously and quantitatively monitored in a label-free and real-time manner. The combined system will give more detailed information of biomolecule/substrate interface for development of new biomaterial.