Quartz Crystal Microbalance Instrument Research Articles

Development of an open-source thermally stabilized quartz crystal microbalance instrument for biomolecule-substrate binding assays on gold and graphene

The interaction of biomolecules, such as proteins, with biomaterial surfaces is key to disease diagnostic and therapeutic development applications. There is a significant need for rapid, low-cost, field-serviceable instruments to monitor such interactions, where open-source tools can help to improve the accessibility to disease screening instruments especially in low- and middle-income countries. We have developed and evaluated a low-cost integrated quartz crystal microbalance (QCM) instrument for biomolecular analysis based on an open-source QCM device. The custom QCM instrument was equipped with a custom-made electronically controlled isothermal chamber with a closed-loop control routine. A thermal coefficient of 5.6 ppm/°C was obtained from a series of evaluations of the implemented control. Additionally, a custom-designed data acquisition system and a mathematical processing and analysis tool is implemented. The quartz crystal detection chips used here incorporate gold and reduced graphene oxide (rGO) coated surfaces. We demonstrate the system capability to monitor and record the biomolecular interaction between a typical protein bovine serum albumin (BSA) and these two substrates. This instrument was compared to a commercial QCM, demonstrating good correspondence between the computed mass adsorption density responses using the Sauerbrey model. For both Au and rGO surfaces, the custom QCM significantly outperforms the commercial system in limit of detection, sensitivity and linear range. The instrument presented here has the potential to serve as a ubiquitous bioelectronic tool for point-of-care disease screening and rapid therapeutics development.

Partitioning of Catechol Derivatives in Lipid Membranes: Implications for Substrate Specificity to Catechol-O-methyltransferase.

We have utilized multiparametric surface plasmon resonance and impendance-based quartz crystal microbalance instruments to study the distribution coefficients of catechol derivatives in cell model membranes. Our findings verify that the octanol–water partitioning coefficient is a poor descriptor of the total lipid affinity for small molecules which show limited lipophilicity in the octanol–water system. Notably, 3-methoxytyramine, the methylated derivative of the neurotransmitter dopamine, showed substantial affinity to the lipids despite its nonlipophilic nature predicted by octanol–water partitioning. The average ratio of distribution coefficients between 3-methoxytyramine and dopamine was 8.0. We also found that the interactions between the catechols and the membranes modeling the cell membrane outer leaflet are very weak, suggesting a mechanism other than the membrane-mediated mechanism of action for the neurotransmitters at the postsynaptic site. The average distribution coefficient for these membranes was one-third of the average value for pure phosphatidylcholine membranes, calculated using all compounds. In the context of our previous work, we further theorize that membrane-bound enzymes can utilize membrane headgroup partitioning to find their substrates. This could explain the differences in enzyme affinity between soluble and membrane-bound isoforms of catechol-O-methyltransferase, an essential enzyme in catechol metabolism.

In situ viscoelastic properties and chain conformations of heavily hydrated carboxymethyl dextran layers: a comparative study using OWLS and QCM-I chips coated with waveguide material

Hydration, viscoelastic properties and dominant structure of thin polymer layers on the surface of waveguide material were evaluated using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance (QCM) methods. The fundamentally different principles of the two applied label-free biosensors enable to examine analyte layers from complementary aspects, e.g. to determine the amount of bound water in hydrated layers. In this study, a new QCM instrument with impedance measurement (QCM-I) is introduced. Its specially designed sensor chips, covered by thin film of waveguide material, supply identical surface as used in OWLS sensors, thus enabling to perform parallel measurements on the same type of surface. Viscoelastic analysis of the measured data was performed by our evaluation code developed in MATLAB environment, using the Voinova’s Voigt-based model. In situ deposition experiments on the ultrathin films of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) were conducted for instrumental and code validation. Additionally, a novel OWLS-QCM data evaluation methodology has been developed based on the concept of combining hydration and viscoelastic data with optical anisotropy results from OWLS measurements. This methodology provided insight into the time-dependent chain conformation of heavily hydrated nano-scaled layers, resulting in unprecedented structural, hydration and viscoelastic information on covalently grafted ultrathin carboxymethyl dextran (CMD) films. The measured mass values as well as hydration and viscoelastic properties were compared with the characteristics of PLL-g-PEG layers.

Self-assembly of bacterial amyloid protein nanomaterials on solid surfaces

HypothesisAmyloid-forming biofilm proteins of Escherichia coli, namely CsgA and CsgB, can form self-assembled nanofibers on solid surfaces. These proteins can be programmed to form bio-nanomaterials for functional applications. ExperimentsIn this study, the assembly of the CsgA and CsgB protein on solid surfaces was investigated in real time using a quartz crystal microbalance instrument with dissipation monitoring. The assembly kinetics of the CsgA and CsgB proteins in various settings on solid surfaces were investigated. Protein nanowires were investigated using electron microscopy. FindingsCsgA protein polymers and CsgB-added CsgA polymers form densely packed biofilm on gold surfaces, whereas CsgB polymers and CsgA-added CsgB polymers form biofilms with high water-holding capacity according to the dissipation data. Electron microscopy images of nanofibers grown on gold surfaces showed that CsgA and CsgB polymers include thicker nanofibers compared to the nanofibers formed by CsgA-CsgB protein combinations. The resulting nano/microstructures were found to have strong fluorescence signals in aqueous environments and in chloroform while conserving the protein nanowire network.

A Highly Sensitive Immunosorbent Assay Based on Biotinylated Graphene Oxide and the Quartz Crystal Microbalance

A high-sensitivity flow-based immunoassay is reported based on a gold-coated quartz crystal microbalance (QCM) chip functionalized directly in the QCM without requiring covalent conjugation steps. Specifically, the irreversible adsorption of a biotinylated graphene oxide-avidin complex followed by loading of a biotinylated capture antibody is applied to avoid more complex conventional surface modification chemistries and enable chip functionalization and sensing all within the QCM instrument. The resulting immunosensors exhibit significantly lower nonspecific protein adsorption and stronger signal for antigen sensing relative to simple avidin-coated sensors. Reproducible quantification of rabbit IgG concentrations ranging from 0.1 ng/mL to 10 μg/mL (6 orders of magnitude) can be achieved depending on the approach used to quantify the binding with simple mass changes used to detect higher concentrations and a horseradish peroxidase-linked detection antibody that converts its substrate to a measurable precipitate used to detect very low analyte concentrations. Sensor fabrication and assay performance take ∼5 h in total, which is on par with or faster than other techniques. Quantitative sensing is possible in the presence of complex protein mixtures, such as human plasma. Given the broad availability of biotinylated capture antibodies, this method offers both an easy and flexible platform for the quantitative sensing of a variety of biomolecule targets.

Small Mass-Change Detectable Quartz Crystal Microbalance and Its Application to Enzymatic One-Base Elongation on DNA

A highly sensitive 27 MHz quartz crystal microbalance instrument with an automatic flow injection system was developed to obtain realistic minimal frequency noise (±0.05 Hz) and to obtain a stable signal baseline (±1 Hz/h) by controlling the temperature of each part in the quartz crystal microbalance (QCM) system using three Peltier devices with a resolution of ±0.001 °C and by optimizing the flow system to prevent fluctuation of the internal pressure of the QCM. The improved QCM with an automatic flow injection system enabled detection of small mass changes such as binding of biotin to a streptavidin-immobilized QCM with a high signal-to-noise ratio. We also applied this device to enzyme reactions of one-base elongation by DNA polymerase (Klenow fragment, KF). We immobilized dsDNAs including the protruding end of dA, dG, dT, or dC on the QCM electrode and ran complementary dNTP monomers with KF into the QCM flow cell. We could directly detect the enzymatic one-base elongation of DNA as a small mass increase, and we found the difference in the reaction rate for each monomer.

QCM sensors for aqueous phenols based on active layers constituted by tetrapyrrolic macrocycle Langmuir films

Three different metalated tetrapyrrolic macrocycles have been transferred by Langmuir-Blodgett technique directly onto piezoelectric quartz crystal covered with gold electrodes of a commercial quartz crystal microbalance instrument in order to perform a flow injection analysis. All floating films at the air-water interface have been analyzed by registration of Langmuir curves and by UV-vis reflection spectroscopy and brewster angle microscopy. The sensing performances of the modified gold electrodes were investigated by monitoring the frequency variation induced by the presence of several toxic phenols. The explored concentrations ranged around 10-3 M and the corresponding frequency variations ranged between 10 and 200 Hz. All responses observed were fast, reproducible and reversible; moreover, the active layers are stable over long periods of utilization. The observation that interferences from fulvic acid are absolutely negligible is also noteworthy. The responses are not selective for each singular phenol derivative; notwithstanding this, to the best of our knowledge, this contribution represents one of the first examples of sensing layers for the monitoring of the total content of phenols.

The adsorption of hyperbranched polymers on silicon oxide surfaces

The electrostatic interaction between quarternised hyperbranched polymers (polyesteramides) and a silicon oxide (SiO 2) surface has been studied via adsorption studies with quartz crystal microbalance instrument with dissipation (QCM-D). Frequency shift (Δ f) results show that the increase in both pH and salt concentration positively affect the adsorbed amount of these polymers, calculated by Sauerbrey equation, on the QCM crystal. The adsorbed amount of HA1 (with lower molecular weight and higher charge density) was lower than that of HA2 (with higher molecular weight and lower charge density) in all experiments. It was also observed that there are no significant changes in the dissipation after adsorption of these polymers. This indicated that both hyperbranched polymers formed rigid adsorbed layers on the negative SiO 2 surface. Additionally, the results were compared with adsorption of poly-DADMAC and the results showed that the hyperbranched polymers formed thicker and more rigid layers as compared with the poly-DADMAC.

Piezoelectric sensor functionalised by a self-assembled bipyridinium derivative: characterisation and preliminary applications in the detection of heavy metal ions

The synthesis of a molecule, 1-(11-dodecylsulfanyl-undecyl)-[4,4′]bipyridinium bromide ( 1), suitable at the same time to form a covalent bond with gold electrodes of a piezoelectric quartz crystal and to interact with heavy metal ions in aqueous solutions, has been successfully accomplished. A commercial quartz crystal microbalance instrument has been modified in order to perform a Flow-Injection Analysis. The behaviour of the system follows the Kanazawa–Gordon equation as demonstrated by measurements with glucose solutions. The self-assembled layer of 1 onto a gold electrode has been characterised by Atomic Force Microscopy before and after the sensing tests. The sensing performances of the modified gold electrode were investigated by monitoring the frequency variation induced by the presence of heavy metal ions, such as lead, cadmium and mercury, in aqueous media. The explored concentrations ranged between 10 −4 and 10 −2 M and the corresponding frequency variations ranged between 10 and 50 Hz. All responses observed were fast, reproducible and reversible. In particular, the response to mercury appears significantly higher in comparison with the other analytes. To the best of our knowledge, this contribution represents the first example of sensing layer based on bipyridinium receptor showing reversible and, at some extent, differentiated response towards heavy metal ions.

Carbon layers in the divertor of ASDEX Upgrade

One of the main disadvantages of carbon as first wall material in a fusion device is the co-deposition of hydrogen with the eroded carbon. These layers will contain a significant amount of the tritium inventory of a fusion reactor. After venting brownish layers and flakes were found under the divertor structure of ASDEX Upgrade. First investigations were made on these flakes. Due to the complicated structure beyond the divertor, shadowing effects occur indicating that the brown layers are deposited by ionised particles. The flakes were analysed using SEM and ion beam techniques. Two different types of hydrocarbon layers were found: The brownish hydrogen poor layer (D/C = 0.4) and transparent hydrogen rich layer (D/C = 1) The total amount of carbon beyond the divertor could be estimated to 1.5 g, deposited in 3000 s of plasma discharges. First measurement of the layer growth using a quartz crystal microbalance instrument is presented.

Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation

The coagulation of blood plasma and whole blood was studied with a surface plasmon resonance (SPR) based device and a quartz crystal microbalance instrument with energy dissipation detection (QCM-D). The SPR and QCM-D response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. The QCM-D response time was longer than SPR, as a physical coupling of the sample to the substrate is required for molecules to be detected by the QCM-method. Change of sample properties within the evanescent field is sufficient for detection with SPR. Both the SPR signals and the QCM-D frequency and dissipation shifts showed dependency on concentrations of coagulation activator and sensitivity to heparin additions. The ratio of dissipation to frequency shifts, commonly considered to reflect viscoelastic properties of the sample, varied with the concentration of activator in blood plasma but not in whole blood. Additions of heparin to the thromboplastin activated whole blood sample, however, made the ratio variation reoccur. Implications of these observations for the understanding of the blood coagulation processes as well as the potential of the two methods in the clinic and in research are discussed.

Scanning Electrochemical Microscopy. 36. A Combined Scanning Electrochemical Microscope−Quartz Crystal Microbalance Instrument for Studying Thin Films

The design of a combined scanning electrochemical microscope-quartz crystal microbalance (SECM-QCM) with separate potential control of the tip and substrate is described. Both lateral and vertical tip movements near the substrate affect the QCM resonant frequency because of perturbations of the longitudinal and shear waves of the quartz crystal (QC) acoustic wave sensor. The SECM-QCM was used to study etching of a thin Ag layer deposited on the QC contact by generating an etchant, iron(III) tris(bipyridine), at the tip near the surface. The SECM-QCM was also used to monitor film mass and surrounding electrolyte composition during potential cycling of a film of C(60) on an electrode.

Energy Dissipation Kinetics for Protein and Antibody−Antigen Adsorption under Shear Oscillation on a Quartz Crystal Microbalance

A new quartz crystal microbalance instrument, allowing simultaneous frequency (f) and dissipation factor (D) measurements, has been used to study protein adsorption kinetics by measuring time-resolved data of both the D-factor, measuring the energy dissipation due to the added overlayer, and the f-shift, measuring the effective mass load on the sensor. Four model proteins (myoglobin, hemoglobin, human serum albumin (HSA), ferritin) and one antibody−antigen reaction (antibody against HSA) were studied on a hydrophobic, methyl-terminated (−CH3) gold surface. In all five cases system-specific, positive D-shifts and negative f-shifts were observed, revealing different adsorption phases. The D-factor measurements provide new information about protein adsorption and improve the interpretation of the frequency shift in terms of mass uptake. Possible mechanisms for the adlayer-induced dissipation are discussed.