Development of a sensitive method for selection of affinity ligand for trypsin using quartz crystal microbalance sensor

In this work, a new methodology is developed for selection of affinity ligands towards the enzyme “trypsin” using quartz crystals microbalance (QCM) technique. To achieve this goal, the surface amination of gold plated QCM crystals was achieved in 13.56 MHz plasma polymerization system by using ethylenediamine. Three different ligands (i.e., 4-aminobenzamidine, 4-aminobenzoic acid, and phenylalanine) were immobilized on the aminated QCM crystals surface via glutaraldhyde coupling. All three ligand immobilized QCM crystals were characterized and compared under different experimental conditions. It was observed that the benzamidine ligand showed higher affinity to trypsin with a dissociation constant on the order of 1.76 × 10⁻⁹ M, which is within the range of 10⁻⁴–10⁻⁸ M for affinity ligands. Thus, its selectivity was suitable for purification of trypsin from biological fluids.     

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.     

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.     

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.     

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.     

Application of parylene-coated quartz crystal microbalance for on-line real-time detection of microbial populations

A novel technique of applying a quartz crystal microbalance (QCM) sensor to the on-line real-time detection of microbial populations is described. The pQCM sensor was fabricated by depositing di-para-xylene (parylene) over the entire surface of a QCM sensor through a chemical vapor deposition (CVD) process. An electrically insulated film of parylene on the QCM sensor enabled the operation of the sensor in the liquid environment, and the resonance frequency of the pQCM sensor set in the medium of a cultivation flask shifted in response to the microbial population. The effects of pH, conductivity, and viscosity of the medium on the frequency shift of the pQCM sensor were investigated. Ignorable responses (less than 1% at 10(3)cells) were obtained during an incubation cycle. The detection limit of the pQCM sensor was identified as 10(2) cells ml(-1) with a frequency shift of around 2 x 10(3)Hz. The cell numbers of Escherichia coli cultivated in both the YEM medium and whole milk were detected. A satisfactory correlation (r(2)=0.95) was obtained between the cell number and the response of the pQCM sensor. Experimental results suggest that the pQCM described here is applicable to the continuous long-term detection of microbial populations during a fermentation process.     

Elongation kinetics of polyglutamine peptide fibrils: a quartz crystal microbalance with dissipation study

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, including Huntington's disease. Expansion of the polyglutamine (polyQ) domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. Studies of synthetic polyQ peptides have contributed substantially to our understanding of the mechanism of aggregation. In this report, polyQ fibrils were immobilized onto a sensor, and their elongation by polyQ peptides of various length and conformation was examined using quartz crystal microbalance with dissipation monitoring (QCM-D). The rate of elongation increased as the peptide length increased from 8 to 24 glutamines (Q8, Q20, and Q24). Monomer conformation affected elongation rates: insertion of a β-turn template d-Pro-Gly in the center of the peptide increased elongation rates several-fold, while insertion of Pro-Pro dramatically slowed elongation. Dissipation measurements of the QCM-D provided qualitative information about mechanical properties of the elongating fibrils. These data showed clear differences in the characteristics of the elongating aggregates, depending on the specific identity of the associating polyQ peptide. Elongation rates were sensitive to the pH and ionic strength of the buffer. Comparison of QCM-D data with those obtained by optical waveguide lightmode spectroscopy revealed that very little water was associated with the elongation of fibrils by the peptide containing d-Pro-Gly, but a significant amount of water was associated when the fibrils were elongated by Q20. Together, the data indicate that elongation of polyQ fibrils can occur without full consolidation to the fibril structure, resulting in variations to the aggregate structure during elongation.     

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.     

Real-time investigation of the muco-adhesive properties of Lactococcus lactis using a quartz crystal microbalance with dissipation monitoring

This work was devoted to probe, at the entire population level, interactions between mucins and Lactococcus lactis, using QCM-D. Real-time monitoring of adsorption on polystyrene of PGM (Pig Gastric Mucin) and subsequent adhesion of L. lactis was performed for IBB477 and MG1820 strains. Measuring simultaneously shifts in resonance frequency and dissipation on the polystyrene-coated crystal demonstrated a two-phase process for PGM adsorption. XPS analysis confirmed the presence of adsorbed mucin. The Voigt-based model was used to describe the QCM-D outputs. The predicted thickness of the PGM layer was consistent with the AFM experimental value. Adhesion of L. lactis to bare or PGM-coated polystyrene was then monitored, in combination with DAPI cell counting. Positive frequency shifts were caused by adhering bacteria. The presence of adsorbed PGM strongly reduced bacterial adhesion. However, adhesion of IBB477 to the PGM coating was greatly increased in comparison with that of MG1820. Muco-adhesion may be a highly variable and valuable phenotypic trait among L. lactis strains.     

Real-time investigation of the muco-adhesive properties of Lactococcus lactis using a quartz crystal microbalance with dissipation monitoring

This work was devoted to probe, at the entire population level, interactions between mucins and Lactococcus lactis, using QCM-D. Real-time monitoring of adsorption on polystyrene of PGM (Pig Gastric Mucin) and subsequent adhesion of L. lactis was performed for IBB477 and MG1820 strains. Measuring simultaneously shifts in resonance frequency and dissipation on the polystyrene-coated crystal demonstrated a two-phase process for PGM adsorption. XPS analysis confirmed the presence of adsorbed mucin. The Voigt-based model was used to describe the QCM-D outputs. The predicted thickness of the PGM layer was consistent with the AFM experimental value. Adhesion of L. lactis to bare or PGM-coated polystyrene was then monitored, in combination with DAPI cell counting. Positive frequency shifts were caused by adhering bacteria. The presence of adsorbed PGM strongly reduced bacterial adhesion. However, adhesion of IBB477 to the PGM coating was greatly increased in comparison with that of MG1820. Muco-adhesion may be a highly variable and valuable phenotypic trait among L. lactis strains.     

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.     

Quantitative measurements of vancomycin binding to self-assembled peptide monolayers on chips by quartz crystal microbalance

This paper describes direct binding of a small vancomycin to peptide ligands immobilized on a sensor chip using quartz crystal microbalance. In this study, the binding ligands were composed of three components: a molecular recognition element (peptide), a conformationally flexible and hydrophilic linker, and a long-chain alkanethiol. These peptide ligands were used to establish the well-packed, self-assembled monolayers on quartz chips and could be readily synthesized using conventional organic chemistry protocols. Results of quartz crystal microbalance measurements showed that vancomycin specifically associated with the d-Ala-d-Ala-containing peptide with an affinity of 3.2+/-0.3 microM and was, as expected, completely inactive to the self-assembled monolayer presenting l-Ala-l-Ala peptide. The dissociation constant obtained correlated well with values reported in literature and was further confirmed by surface plasmon resonance measurement (2.7+/-0.7 microM). The technique used in this study should be applicable to both peptidyl and nonpeptidyl ligands of greater complexity than that used here. This method is practical, it provides quantitative binding information, and complicated analysis is avoided.     

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.     

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.     

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.     

Application of a quartz crystal microbalance to evaluate biodegradability of starch by Bacillus subtilis

Biodegradation of solution-cast starch films by Bacillus subtilis was monitored using a quartz crystal microbalance (QCM). A starch film was formed on the crystal by solution casting and exposed to the Bacillus subtilis culture in a bioreactor. The high sensitivity of the QCM could monitor small weight changes of the starch films on the crystal in the initial stages of biodegradation by secreted exo-enzymes of the bacterium. The feasibility of this approach as a means of quantification and characterisation of biodegradability of different polymeric materials by selected organisms is discussed.     

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.     

Covalent DNA immobilization on polymer-shielded silver-coated quartz crystal microbalance using photobiotin-based UV irradiation.

The use of a commercial, silver-coated quartz crystal microbalance (QCM) as a disposable, low-cost, and reliable DNA sensor is presented. This is an incorporation of polymer-based silver electrode shielding and photochemistry-based surface modification for covalent DNA immobilization. To prevent undesired oxidation, the silver electrodes are coated with thin polystyrene films. The polymer surfaces are then modified by a photoreactive biotin derivative (photobiotin) under UV irradiation. The resulting biotin residues on the polymer-shielded surface react with a tetrameric avidin. Consequently a biotin-labeled DNA probe can be immobilized through a biotin-avidin-biotin bridge. A 14-mer single-stranded biotin-DNA probe and a 70-mer single-stranded DNA fragment containing complementary or noncomplementary sequences are used as a model system for DNA hybridization assay on the proposed sensors. The shielding ability of the polystyrene coatings after photo irradiation is investigated. The DNA probe binding capacity, hybridization efficiency, and kinetics are also investigated.     

Study on colloidal Au-enhanced DNA sensing by quartz crystal microbalance

Colloidal Au is reported for enhancement the immobilization capacity and ultimately detection limit of DNA using quartz crystal microbalance (QCM). Immobilization of approximately 12 nm-diameter colloidal Au on to an Au-coated QCM resulted in an easier attachment of oligonucleotide, with a mercaptohexyl group at the 5'-phosphate end and an increased capacity for nucleic acid detection. DNA immobilization and hybridization was monitored from QCM frequency changes. Hybridization was induced by exposure of the DNA-containing films to complementary DNA in solution. A much higher sensitivity was obtained for the analyte. The Au nanoparticle films on the Au plate provide a novel means for the fabrication of DNA sensor.     

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.