Electroimmobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purification

Proinsulin C-peptide was electroimmobilized to a quartz crystal microbalance sensor chip, localizing this low-pI peptide for covalent attachment to activated surface carboxyl groups. The resulting chip was used in a continuous flow biosensor to capture anti-C-peptide antibodies, which could subsequently be eluted in 5% formic acid between air bubbles for efficient recovery and mass spectrometric identification. The method is reproducible through repeated cycles, providing affinity purification of proteins under real-time monitoring of the binding and elution processes.     

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.     

A biosensor for estrogenic substances using the quartz crystal microbalance

This article describes a biosensor that detects estrogenic substances using a quartz crystal microbalance with a genetically engineered construct of the hormone-binding domain of the alpha-estrogen receptor. The receptor was immobilized to a piezoelectric quartz crystal via a single exposed cysteine, forming a uniform orientation on the crystal surface. Our results illustrate that this sensor responds to a variety of ligands that are known to bind to the estrogen receptor. No response was observed for nonbinding substances such as testosterone and progesterone. The sensitive response of this biosensor to estrogenic substances results from changes in the structural rigidity of the immobilized receptor that occurs with ligand binding. Agonist and antagonist show different responses.     

Oligonucleotide quartz crystal microbalance sensor for the microalgae Alexandrium minutum (Dinophyceae)

We report the immobilization on a gold surface of a 20-base DNA probe labeled with disulfide group and on the selective hybridization with the complementary 20-base DNA strand. The oligonucleotide probe is the complementary strand of a partial sequence of the gene encoding for a large ribosomal RNA sub-unit which is a coding sequence of Alexandrium minutum DNA, a microalgae that produces neurotoxins responsible for paralytic shellfish poisoning on European and Asian coasts. The kinetics of DNA probe immobilization and hybridization were monitored in situ by using a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the setup is stable to within a few hertz, corresponding to the nanogram scale, for 3h and makes it possible to follow frequency change from immobilization of the probe to hybridization of the complementary DNA target. This setup constitutes a biosensor, which is sensitive and selective, and the hybridization ratio between hybridized complementary DNA and immobilized DNA probes is 47%.     

Specific and selective peptide-membrane interactions revealed using quartz crystal microbalance

The skin secretions of Australian tree frogs are rich in peptides with potential antimicrobial activity. They interrupt bacterial cell membranes, although precisely how and whether all peptides have the same mechanism is not known. The interactions of three of these peptides—aurein 1.2, maculatin 1.1, and caerin 1.1 with supported phospholipid bilayers—are examined here using quartz crystal microbalance and atomic force microscopy. These approaches enabled us to reveal variations in material structure and density as a function of distance from the sensor surface when comparing mass sensorgrams over a range of harmonics of the natural resonance of the sensor crystal and hence obtain for the first time to our knowledge a mechanistic assessment of membrane disruption. We found that caerin inserted into the bilayer in a transmembrane manner, regardless of concentration and phospholipid composition consistent with a pore-forming mechanism. In contrast, maculatin and aurein interacted with membranes in a concentration-dependent manner. At low concentrations (<5 μM), maculatin exhibited transmembrane incorporation whereas aurein was limited to surface association. Upon reaching a threshold value of concentration, both peptides lysed the membrane. In the case of maculatin, the lysis progressed in a slow, concentration-dependent manner, forming mixed micelles, as shown by atomic force microscopy imaging. Aurein-induced lysis proceeded to a sudden disruption, which is consistent with the “carpet” mechanism. Both maculatin and aurein exhibit specificity toward phospholipids and thus have potential as candidates as antimicrobial drugs.     

Self‐assembly and chiral recognition of quartz crystal microbalance chiral sensor

A novel chiral sensor based on the self‐assembled monolayer of (6^A‐ω‐mercaptoethylureado‐6^A‐deoxy)heptakis(2,3‐di‐o‐phenylcarbamoyl)‐6^B, 6^C, 6^D, 6^E, 6^F, 6^G‐ hexa‐o‐phenylcarbamoyl‐β‐cyclodextrin (Ph‐β‐CD‐SH) on a quartz crystal transducer for chiral recognition was set up. (R,S)‐(±)‐(3‐Methoxyphenyl)ethylamine were recognized by this QCM chiral sensor with a QCM chiral discrimination factor of 1.33. Furthermore, UV spectroscopy was used to investigate the mechanism of host‐guest interactions between (6^A‐azido‐6^A‐deoxy)heptakis(2,3‐di‐o‐phenylcarbamoyl)‐6^B, 6^C, 6^D, 6^E, 6^F, 6^G‐hexa‐o‐phenylcarbamoyl‐β‐cyclodextrin (Ph‐β‐CD) and (R,S)‐(±)‐(3‐methoxyphenyl) ethylamine. The UV discrimination factor was determined to be 0.066. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

A quartz crystal microbalance method for rapid detection and differentiation of shiga toxins by applying a monoalkyl globobioside as the toxin ligand

A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method. The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film. Then, the film was transferred onto a QCM cell surface modified with alkanethiols. Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell. Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins. On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer.     

Human cytomegalovirus detection by a quartz crystal microbalance immunosensor

A piezoelectric affinity sensor has been developed to detect distinctive antigens of the human cytomegalovirus. Either the specific antibodies or the antigen were immobilized on the gold electrode. To develop a rapid immunoassay, various assay formats were tested in relation with the different antigen composition. First, a direct assay was carried out immobilizing the specific antibody on the crystal surface by passive adsorption. Next, Protein A, thiol/poly L-lysine mixed self-assembled monolayers were tested as methods of gold modification. A competitive format was exploited by immobilization of the antigen onto the crystal activated by SAM and poly L-lysine. This procedure yielded a preliminary calibration curve. A linear range between 2.5 and 5 μg/ml of gB epitope in solution and a detection limit of 1 μg/ml were measured.     

Monitoring the conformational changes of an intrinsically disordered peptide using a quartz crystal microbalance

Intrinsically disordered peptides (IDPs) have recently garnered much interest because of their role in biological processes such as molecular recognition and their ability to undergo stimulus-responsive conformational changes. The block V repeat-in-toxin motif of the Bordetella pertussis adenylate cyclase is an example of an IDP that undergoes a transition from a disordered state to an ordered beta roll conformation in the presence of calcium ions. In solution, a C-terminal capping domain is necessary for this transition to occur. To further explore the conformational behavior and folding requirements of this IDP, we have cysteine modified three previously characterized constructs, allowing for attachment to the gold surface of a quartz crystal microbalance (QCM). We demonstrate that, while immobilized, the C-terminally capped peptide exhibits similar calcium-binding properties to what have been observed in solution. In addition, immobilization on the solid surface appears to enable calcium-responsiveness in the uncapped peptides, in contrast to the behavior observed in solution. This work demonstrates the power of QCM as a tool to study the conformational changes of IDPs immobilized on surfaces and has implications for a range of potential applications where IDPs may be engineered and used including protein purification, biosensors, and other bionanotechnology applications.     

Amplified detection of telomerase activity using electrochemical and quartz crystal microbalance measurements

Telomerase is considered as an important biomarker for cancer cells. Two different methods for the amplified electrochemical and microgravimetric quartz-crystal-microbalance detection of telomerase activity originating from HeLa cancer cells are described. One method involves the telomerization of a primer (1) linked to the electrode, in the presence of telomerase from HeLa cell extract and dNTP, followed by the hybridization of a biotin-labeled nucleic acid (2) that is complementary to the telomere repeat units. The subsequent binding of an avidin-alkaline phosphatase conjugate (3) that catalyzes the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (4) results in the precipitation of the insoluble product (5) on the electrode. The second method involves the telomerization of the primer (1) associated with the electrode, in the presence of the telomerase-containing HeLa cell extract and the dNTP nucleotide mixture that includes biotin-labeled dUTP. The telomerization leads to the labeling of the telomeres with biotin labels. The association of the avidin-alkaline phosphatase conjugate (3) to the biotin labels results in the biocatalyzed transformation of (4) to (5) and the formation of a precipitate on the electrode or the Au-quartz crystal. As numerous precipitate molecules are formed as a result of the formation of a single telomere, the methods represent routes for the amplified detection of telomerase activity. The formation of the precipitate on the respective transducers is probed by following the changes in the electrode resistance using chronopotentiometry, or by following the frequency changes of the piezoelectric quartz crystals. The amount of precipitate generated on the electrodes is controlled by the concentration of the HeLa cancer cells. The methods enable the detection of telomerase activity that is extracted from 1000 HeLa cancer cells.     

Chiral discrimination using a quartz crystal microbalance and comparison with gas chromatographic retention data

Quartz crystal microbalances (QCMB) have been constructed using 10 MHz AT cut quartz crystals coated with heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, heptakis(6-O-methyl-2,3-di-O-pentyl)-β-cyclodextrin, and octakis(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as 50% and 20% (w/w) solutions in OV1701. The reduction in frequency seen on exposure of each coated QCMB to pure enantiomeric forms of α- and β-pinene and cis- and trans-pinane show that statistically significant (P = 0.05, n = 7) differences are observed between the enantiomeric pairs. The apparent preferential binding shown by the QCMB for enanciomers of α- and β-pinene and cis- and trans-pinane have been compared with the elution order observed on the corresponding gas chromatographic stationary phase. The magnitude of the observed separation factor (calculated as the ratio of the OV1701 normalised frequency shift) is seen to be dependent upon the chiral stationary phase concentration. These results indicate that on-line determination of enantiomeric excess and concentration of certain monoterpenes is possible at room temperature using QCMB in conjunction with chiral gas chromatographic stationary phases. Chirality 9:225–232, 1997. © 1997 Wiley-Liss, Inc.     

Real-time molecular recognition between protein and photosensitizer of photodynamic therapy by quartz crystal microbalance sensor

Real-time investigation of molecular recognition between protein and the photosensitizer of photodynamic therapy (PDT) was carried out by a quartz crystal microbalance (QCM) sensor integrated into a flow injection analysis (FIA) system. The photosensitizer meso-tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) was immobilized on the gold electrode of the QCM chip by combining the sol-gel and self-assembly methods. Such a rapid screen analysis of molecular recognition showed that the p-THPP-immobilized sensor exhibited sensitive and specific interaction only with hemoglobin (Hb). The kinetic rate constants (k_ass and k_diss) and the equilibrium association constant (K_A) for p-THPP-Hb interaction were calculated by linear regression. The sensing performance characteristics of the proposed sensor were investigated. The sensor showed excellent selectivity, reproducibility, and repeatability for the detection of Hb. A linear calibration plot was obtained over a range from 0.2 to 1.0 μM with a detection limit (signal/noise ratio = 3) of 0.15 μM. The response mechanism of the sensor is discussed in detail. Due to its low cost and simple manipulation, this QCM-FIA system was shown to be a highly effective method for the investigation of interaction between biomacromolecules and the PDT photosensitizer. It also provides a potential strategy for screening an efficient and less harmful photosensitizer for PDT application.     

Long-term monitoring of biofilm growth and disinfection using a quartz crystal microbalance and reflectance measurements

A biofilm reactor was constructed to monitor the long-term growth and removal of biofilms as monitored by the use of a quartz crystal microbalance (QCM) and a novel optical method. The optical method measures the reflectance of white light off the surface of the quartz crystal microbalance electrode (gold) for determination of the biofilm thickness. Biofilm growth of Pseudomonas aeruginosa (PA) on the surface was used as a model system. Bioreactors were monitored for over 6 days. Expressing the QCM data as the ratio of changes in resistance to changes in frequency (DeltaR/Deltaf) facilitated the comparison of individual biofilm reactor runs. The various stages of biofilm growth and adaptation to low nutrients showed consistent characteristic changes in the DeltaR/Deltaf ratio, a parameter that reflects changes in the viscoelastic properties of the biofilm. The utility of white light reflectance for thickness measurements was shown for those stages of biofilm growth when the solution was not turbid due to high numbers of unattached cells. The thickness of the biofilms after 6 days ranged from 48 mum to 68 mum. Removal of the biofilm by a disinfectant (chlorine) was also measured in real time. The combination of QCM and reflectance allowed us to monitor in real time changes in the viscoelastic properties and thickness of biofilms over long periods of time.     

Optimisation of the enzyme-based determination of hydrogen peroxide using the quartz crystal microbalance

The benzidines, 3,3'-diaminobenzidine (DAB), 3,3'-dimethoxybenzidine (DMOB) and 3,3',5,5'-tetramethylbenzidine (TMB) were enzymatically oxidised to detect hydrogen peroxide, using the quartz crystal. The oxidised product mainly remains in suspension, resulting in a limited quartz sensor signal. We have used two non-ionic surfactants, Tween 80 and Triton X-100 to interact with the oxidised amphiphilic products to increase their solubility and surface activity, and their ability to adsorb to the crystal surface. Tween 80 exhibits optimised response effects for DAB, DMOB and TMB at 0.012, 0.005, and 0.002% (v/v), respectively, whereas Triton X-100 is optimum at 0.1, 0.2, and 0.006% (v/v), respectively. As a result, we have improved the quartz crystal sensor sensitivity to peroxide. The use of Triton X-100 gave an improved response time.     

Sensing HIV related protein using epitope imprinted hydrophilic polymer coated quartz crystal microbalance

We have developed a biomimetic sensor for the detection of human immunodeficiency virus type 1 (HIV-1) related protein (glycoprotein 41, gp41) based on epitope imprinting technique. gp41 is the transmembrane protein of HIV-1 and plays an important role in membrane fusion between viruses and infected cells. It is an important index for determining the extent of HIV-1 disease progression and the efficacy of therapeutic intervention. In this work, dopamine was used as the functional monomer and polymerized on the surface of quartz crystal microbalance (QCM) chip in the presence of template, a synthetic peptide with 35 amino acid residues, analogous to residues 579-613 of the gp41. This process resulted in grafting a hydrophilic molecularly imprinted polymer (MIP) film on the QCM chip. QCM measurement showed that the resulting MIP film not only had a great affinity towards the template peptide, but also could bind the corresponding gp41 protein specifically. The dissociation constant (K(d)) of MIP for the template peptide was calculated to be 3.17 nM through Scatchard analysis, which was similar to those of monoclonal antibodies. Direct detection of the gp41 was achieved quantitatively using the resulting MIP-based biomimetic sensor. The detection limit of gp41 was 2 ng/mL, which was comparable to the reported ELISA method. In addition, the practical analytical performance of the sensor was examined by evaluating the detection of gp41 in human urine samples with satisfactory results.     

A survey of the 2010 quartz crystal microbalance literature

In 2010 there has again been an increase in the number of papers published involving piezoelectric acoustic sensors, or quartz crystal microbalances (QCM), when compared to the last period reviewed 2006‐2009. The average number of QCM publications per annum was 124 in the period 2001‐2005, 223 in the period 2006‐9, and 273 in 2010. There are trends towards increasing use of QCM in the study of protein adsorption to surfaces (93% increase), homeostasis (67% increase), protein‐protein interactions (40% increase), and carbohydrates (43% increase). New commercial systems have been released that are driving the uptake of the technology for characterisation of binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights theoretical and practical aspects of the principals that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells, and membrane interfaces. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a quartz crystal microbalance (QCM) immunosensor for the detection of Listeria monocytogenes

This study presents the development of a QCM immunosensor for the detection of Listeria monocytogenes. A self-assembled monolayer (SAM) of thiosalicylic acid is incorporated for the covalent attachment of antibodies to the gold surface of the piezoelectric crystal. A non-Sauerbrey increase in frequency is observed upon exposure of such a crystal to specific antigen cells. This unexpected response is consistent with the obtained results and is shown to be specific. The sensor can detect L. monocytogenes cells in real time in solution to 1 × 10⁷ cells/ml. The sensor is reusable more than 10 times without detectable loss in activity and shows negligible response to a non-specific pathogen, Bacillus cereus. The lifetime of the thiolated crystal was also investigated.     

Cell adhesion monitoring using a quartz crystal microbalance: comparative analysis of different mammalian cell lines

The quartz crystal microbalance (QCM) has been widely accepted as a sensitive technique to follow adsorption processes in gas as well as in liquid environments. However, there are only a few reports about the use of this technique to monitor the attachment and spreading of mammalian cells onto a solid support in culture. Using a QCM-setup we investigated the time course of cell attachment and spreading as a function of seeding density for three widespread and frequently used cell lines (MDCK strains I and II and Swiss 3T3-fibroblasts). Results were found to be in good agreement with the geometrical properties of the individual cell types. The shifts of the resonance frequency associated with confluent cell layers on top of the quartz resonators were found to be dependent on the cell species [MDCK-I: (320±20) Hz; MDCK-II: (530±25) Hz; 3T3: (240±15) Hz] reflecting their individual influence on the shear oscillation of the resonator. These findings are discussed with respect to the basic models of materials in contact with an oscillating quartz resonator. We furthermore showed by inhibition-assays using soluble RGD-related peptides, that only specific, integrin mediated cell adhesion is detected using this QCM approach, whereas the sole presence of the cellular body in close vicinity to the resonator surface is barely detectable.     

Real-time monitoring of the cell agglutination process with a quartz crystal microbalance

The real-time monitoring of the agglutination process of human hepatic normal cells (L-02) at the quartz crystal microbalance (QCM) gold (Au) electrode was performed. Two lectins, concanavalin A (Con A) and wheat germ agglutinin (WGA), induced the cell agglutination, resulting in the different Δf₀ and ΔR₁ responses from those caused by the normal cell attachment and growth. The cell-Con A-cell aggregates had higher affinity for the Au substrate due to the excellent adsorption ability of Con A, which was revealed by increased Δf₀ and ΔR₁ shifts and the obvious mass effect of QCM. In contrast, the lower adsorption ability of cell-WGA-cell aggregates was related to the same characteristic of WGA, presenting the decreased Δf₀ and ΔR₁ responses and the time-extended adhesion phase. Parallel microscopic observation experiments were also carried out and exhibited comparable results. The Δf₀ responses during the processes of cell growth and cell agglutination were analyzed using the equations Δf₀=a₀+a₁e^-t/τ₁+a₂e^-t/τ₂+a₃e^-t/τ₃ and Δf₀=a₀+a₁e^-t/τ₁+a₂e^-t/τ₂, respectively. Furthermore, the current work proved that the QCM measurement technique based on cell agglutination was useful for discriminating hepatic normal cells (L-02) and hepatic cancer cells (Bel7402).     

A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7

A quartz crystal microbalance (QCM) DNA sensor, based on the nanoparticle amplification method, was developed for detection of Escherichia coli O157:H7. A thiolated single-stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene was immobilized onto the QCM sensor surface through self-assembly. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA, and resulted in the mass change and therefore frequency change of the QCM. Streptavidin conjugated Fe(3)O(4) nanoparticles (average diameter=145 nm) were used as "mass enhancers" to amplify the frequency change. Synthesized biotinylated oligonucleotides as well as E. coli O157:H7 eaeA gene fragments (151 bases) amplified using asymmetric PCR with biotin labeled primers were tested. As low as 10(-12)M synthesized oligonucleotides and 2.67 x 10(2) colony forming unit (CFU)/ml E. coli O157:H7 cells can be detected by the sensor. Linear correlation between frequency change and logarithmic number of bacterial cell concentration was found for E. coli O157:H7 from 2.67 x 10(2) to 2.67 x 10(6)CFU/ml.