Detection of specific DNA from crude extracts of rice seed grains using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

In this work, it is shown that the quartz crystal microbalance (QCM) can be a powerful and simple tool for quick and precise kinetic enzymatic assays. This is shown by measuring immobilized acetylcholinesterase (AChE) activity with variations of pH as a case study.     

Acoustic detection of cell adhesion to a coated quartz crystal microbalance – implications for studying the biocompatibility of polymers

Biocompatibility of polymers is an important parameter for the successful application of polymers in tissue engineering. In this work, quartz crystal microbalance (QCM) devices were used to follow the adhesion of NIH 3T3 fibroblasts to QCM surfaces modified with fibronectin (FN) and poly-D -lysine (PDL). The variations in sensor resonant frequency (Δf) and motional resistance (ΔR), monitored as the sensor signal, revealed that cell adhesion was favored in the PDL-coated QCMs. Fluorescence microscopy images of seeded cells showed more highly spread cells on the PDL substrate, which is consistent with the results of the QCM signals. The sensor signal was shown to be sensitive to extracellular matrix (ECM)-binding motifs. Ethylenediaminetetraacetic acid (EDTA) and soluble Gly-Arg-Gly-Asp-Ser (GRGDS) peptides were used to interfere with cell-ECM binding motifs onto FN-coated QCMs. The acquired acoustic signals successfully showed that in the presence of 30 mM EDTA or 1 mM GRGDS, cell adhesion is almost completely abolished due to the inhibition/blocking of integrin function by these compounds. The results presented here demonstrate the potential of the QCM sensor to study cell adhesion, to monitor the biocompatibility of polymers and materials, and to assess the effect of adhesion modulators. QCM sensors have great potential in tissue engineering applications, as QCM sensors are able to analyze the biocompatibility of surfaces and it has the added advantage of being able to evaluate, in situ and in real time, the effect of specific drugs/treatments on cells.     

QCM 监测自组装膜上心肌细胞黏附及其与心血管药物作用

目的:本试验采用石英晶体微天平(QCM)实时监测大鼠心肌细胞(H9C2)在含有细胞黏附识别多肽RGD自组装膜上的动态黏附过程及随后与两种心血管药物(一种正性肌力、另一种负性肌力)相互作用。方法:在金电极表面自组装3-巯基丙酸(MPA)单层膜,并经酰胺化共价耦合细胞黏附分子KRGD,形成对大鼠心肌细胞有特异性黏附的致密分子自组装膜。QCM以动态持续的方式实时监测MPA/RGD自组装及其不同浓度梯度H9C2细胞在自组装膜金电极上的细胞黏附过程。此外,选用20,000个H9C2细胞和正性肌力药物异丙肾上腺素、负性肌力药物维拉帕米,用QCM评估了细胞-心血管药物的相互作用。结果:与裸金电极相比,MPA/RGD修饰金电极增大了H9C2细胞黏附所引起的QCM频移(△f)与动态电阻变化(△R)响应。在所试H9C2浓度范围(5×10~4-4×10~5 cells/m L),△f与H9C2浓度呈线性关系,△R与H9C2浓度呈幂函数关系。我们用细胞粘弹性指数(CVI=△R/△f)来表征细胞的粘弹性。H9C2在异丙肾上腺素作用下,△f与△R增加、细胞-QCM表面黏附加强,细胞变硬;在维拉帕米作用下,△f与△R降低、细胞QCM表面黏附减弱,细胞变软。结论:QCM可用于不同浓度大鼠心肌细胞的动态细胞黏附监测,并可基于其细胞黏附与细胞黏弹性测定能力区分正性与负性肌力药物而可望用于心血管药物的筛选。     

Analysis of the interactions of ribonuclease inhibitor with kanamycin

The interaction of ribonuclease inhibitor (RI) with kanamycin was studied by molecular modeling. The preliminary binding model was constructed using the Affinity module of the Insight II molecular modeling program and the key residues involved in the combination of RI binding to kanamycin were determined. Meanwhile, we determined relevant surface characteristics determining the interaction behavior. The modeling results indicated that electrostatic interactions and H-bond forces may work as major factors for the molecular interaction between kanamycin and RI. The above results are useful for elucidating the molecular principles upon which the selectivity of a kanamycin is based. The quartz-crystal microbalance (QCM) is a new method usually used to monitor the binding function of macromolecules with samples online in a flow-injection analysis (FIA) system. The experimental results demonstrate that kanamycin has an extraordinary affinity to the basic protein RI, and our result is consistent with the molecular modeling results. These principles can in turn be used to study the molecular recognition mechanism and design a mimic of kanamycin for the development of new RI binders.Figure Proposed binding model of kanamycin to RI obtained by the computer-aided docking method and optimized with molecular mechanics with the CVFF forcefield.     

Double-mode impedance analysis of epithelial cell monolayers cultured on shear wave resonators

The viscoelastic behavior of epithelial cells (MDCK-I and MDCK-II) grown on AT-cut quartz crystals with a fundamental resonance at 5 MHz was investigated by impedance spectroscopy. Using the electromechanical model recently derived by Martin et al. [(1991) Anal Chem 63: 2272 – 2281] for Newtonian liquids in contact with shear wave resonators we quantified the viscous damping arising from the adherent cells by fitting the impedance data with a modified Butterworth-Van Dyke circuit in the region of the resonance frequency. Impedance spectroscopy was additionally performed in the frequency range from 1 Hz to 1 MHz to scrutinize the passive electrical properties of the epithelial cell layers using an additional platinum electrode. These data allow one to document the cell layers' integrity as well as the electrode coverage. We were able to confirm that the presence of a cell-layer mainly increases damping of the shear wave and does not exhibit a pure mass-load behavior. These findings were supported by the discovery that the inductance L in the electromechanical model was less influenced by the cell-layer than the resistance R. The apparent cell-viscosities determined by our method are 0.097 poise for MDCK-I and 0.142 poise for MDCK-II cell-layers. These low apparent viscosities may be explained in terms of a considerable spacing between the cells immobilized via their focal contacts and the quartz surface. Received: 5 June 1996 / Accepted: 6 August 1996     

Label free and amplified detection of cancer marker EBNA-1 by DNA probe based biosensors

Epstein-Barr virus (EBV) is a human herpes virus that has been associated with several malignancies as Burkitt's lymphoma, nasopharyngeal carcinoma and Hodgkin's disease. All EBV associated malignancies showed a distinct viral gene expression pattern, while Epstein-Barr nuclear antigen 1 (EBNA-1) is constitutively expressed in all such disorders. Here, the development of a biosensor to detect EBNA-1 protein is reported, which was based on a nucleic acid bioreceptor and a quartz crystal microbalance with a dissipation monitoring (QCM-D) transducer. The DNA probe for EBNA-1 detection was designed and synthesized to mimic its palindromic target sites in the EBV genome. This DNA probe was immobilized on the Au-surface of a QCM-D electrode, followed by the blocking of the accessible Au-surface with 6-mercapto-1-hexanol (6-MHO). The system showed a limit of detection of 50 ng/mL in direct detection of EBNA-1, however, the sensitivity was improved by 2 orders of magnitude (0.5 ng/mL) when an amplification cascade, employing antibodies labeled with alkaline phosphatase (AP), was applied to the system.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

In this study, we describe a detection system for the indirect detection of vaccinia virus by DNA analysis. The system uses quartz crystal microbalance (QCM) as the detection technique and polymerase chain reaction (PCR) for amplification. Different immobilization strategies for the capture probe on the quartz chip are studied. For the QCM detection of hybridisation, the influence of the structure and length of target DNA is analyzed. For the detection of DNA from an amplification product, an efficient denaturation procedure is developed. On the basis of these investigations, vaccinia virus DNA is detected with only a low number of amplification rounds and a short analysis time. Specificity can be clearly shown. To enhance the signal strength and to have a further proof of specificity, a gold nanoparticle-tagged enhancer sequence can be used.     

Designing of MIP based QCM sensor having thymine recognition sites based on biomimicking DNA approach

Quartz crystal microbalance (QCM) sensors coated with molecular imprinted polymers (MIP) have been developed for the determination of thymine. In this method, methacryloylamidoadenine (MA-Ade) have used as a new monomer and thymine template for inspiration of DNA nucleobases interaction. The thymine can be simultaneously hydrogen binding to MA-Ade and fit into the shape-selective cavities. Thus, the interaction between nucleobases has an effect on the binding ability of the QCM sensors. The binding affinity of the thymine imprinted sensors has investigated by using the Langmuir isotherm. The thymine imprinted QCM electrodes have shown homogeneous binding sites for thymine (K_a: 1.0 × 10⁵ M⁻¹) while heterogeneous binding sites for uracil. On the other hand, recognition selectivity of the QCM sensor based on thymine imprinted polymer toward to uracil, ssDNA and ssRNA has been reported in this work.     

Recombinant antibody fragments allow repeated measurements of C-reactive protein with a quartz crystal microbalance immunosensor

C-reactive protein (CRP) is a serum marker highly upregulated in inflammation after bacterial infection. Robust, reliable and quick quantification of CRP would be a substitute for erythrocyte sedimentation rate (ESR) with superior diagnostic value. Quartz crystal microbalance (QCM) based sensors coated with specific antibodies and integrated into lab-on-chip systems are in development for rapid point of care quantification. In this study, we isolated three CRP specific single chain (sc)Fv antibody fragments using phage display from an antibody gene library. Their affinities ranged from 2.7 × 10^?8 to 1.0 × 10^?8 M when measured by surface plasmon resonance. ScFv antibody fragment LA13-IIE3 showed best affinity, high long-term stability and remarkable resistance to denaturation. This scFv antibody fragment was coupled to a QCM sensor. CRP quantification in up to 15 samples sequentially measured on the same sensor with intermitting regeneration by buffer was demonstrated.     

Integrating the QCM detection with magnetic separation for on-line analysis

We investigate the feasibility of coupling the quartz crystal microbalance (QCM) with magnetic separation for on-line analysis. A flow cell was integrated with QCM and magnetic force for the analysis of magnetic and nonmagnetic samples. The resonant frequency change (Deltaf) of QCM was related to the amount of deposited magnetic nanoparticles. This experiment demonstrates that QCM can be used as an on-line detector for magnetic separation. The QCM also gives a characteristic response of the binding between the streptavidin and biotin labeled on the magnetic nanoparticles. Biotin-labeled magnetic nanoparticles were flowed through a gold electrode of QCM to deposit as a matrix for selective capturing streptavidin. The resonant frequency change of QCM was proportional to the amounts of streptavidin captured by biotin. This technique can provide a simple, economic, and automatic method for on-line detection of biomarkers.     

Synthesis and decoloring properties of sodium humate/poly (N-isopropylacrylamide) hydrogels

A series of novel sodium humate/poly(N-isopropylacrylamide) (SH/PNIPA) hydrogels were synthesized by solution polymerization. The swelling and decoloring properties of SH/PNIPA hydrogels were also examined. Experiment results show that there exist hydrogen-bonding interactions between SH and PNIPA in the SH/PNIPA hydrogels network, which are not strong enough to disrupt the aggregation of dehydrated PNIPA chains at phase transition temperature, leading to the same volume phase transition temperature as pure PNIPA hydrogel. The adsorption and desorption of methylene blue (MB) for the hydrogels were influenced by temperature, initial MB concentration and SH amount. Low temperature favors the adsorption and desorption of MB. Appropriate SH amount of the hydrogels is crucial for the adsorption and desorption of MB. The maximum adsorption capacity was 10.8 mg MB per gram of SH/PNIPA gel.     

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.     

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.     

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.     

A biotin-hydrogel-coated quartz crystal microbalance biosensor and applications in immunoassay and peptide-displaying cell detection

A biotin-coated quartz crystal microbalance (QCM) chip was prepared by dip-coating a long-chain alkanethiol-modified crystal with precoupled dextran-biotin hydrogels. The resulting biotin chip was used to affinity-immobilize streptavidin (SAv) and was then further employed for various biosensor assays. First, the SAv chip allowed efficient on-line binding of biotinylated bovine serum albumin (bBSA), followed by a sensitive and specific response toward anti-bovine serum albumin (BSA) antibodies. Three consecutive immunoassays were reproducibly demonstrated with a single chip. The apparent binding kinetics with k_on = 5.9 μM⁻¹ h⁻¹, k_off = 10.1 h⁻¹, and K_D = 1.71 μM was readily resolved by fitting the real-time sensorgrams. Second, the capability of the SAv chip to selectively recognize recombinant Escherichia coli with flagella displaying an artificial SAv binding peptide, Strep-tag II, was demonstrated by QCM analysis and verified by scanning transmission electron microscope (STEM) image analysis with biotin-coated gold nanoparticles as the label. Finally, the affinity of the cell-displayed Strep-tag II peptide to surface-coated SAv, K_D = 6.8 × 10⁸ CFU/ml, was resolved on-line using equilibrium binding kinetics by QCM. This study presents an easy, economical, and reliable method of preparing high-performance SAv-coated biotin chips with potential for application in real-time repetitive immunoassays, on-line binding kinetics studies, and high-affinity peptide screening.     

A novel microgravimetric DNA sensor with high sensitivity

A novel method using an amplifier with a cantilever and gold nanoparticles successfully to extend the length of the target for the specific and high sensitive detection of DNA was reported. When the size of gold nanoparticle is 50 nm, a sensitivity of 10(-15)M for the single base mutation detection has been achieved.     

The enhancement effect of gold nanoparticles as a surface modifier on DNA sensor sensitivity

The enhancement of a single strain DNA probe linked to the sensor surface is of crucial importance in DNA molecule recognition. By means of nanogold modification of the sensor surface in addition to the nanogold amplifier, DNA detection sensitivity higher than 10(-16)mol/L was obtained in a Quartz Crystal microbalance (QCM) system, much higher than the ordinary QCM sensor without surface modification by nanogold.     

Specific binding of peanut agglutinin to GM1-doped solid supported lipid bilayers investigated by shear wave resonator measurements

This study deals with the specific interaction between the lectin peanut agglutinin (PNA) from Arachis hypogaea and the ganglioside G_M1 which was incorporated in a solid supported lipid bilayer immobilized on a gold electrode placed on top of an AT-cut quartz crystal. Bilayer formation was reached by self-assembly processes. The first monolayer consists of octanethiol attached to the gold surface via chemisorption and the second monolayer was immobilized by vesicle fusion on the preformed hydrophobic surface. We managed to keep unspecific binding to a minimum by using a phospholipid matrix consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Lectin binding to ganglioside G_M1 containing membranes was determined by a decrease of the resonant frequency of the quartz crystal. The minimum amount of receptor within the membrane which is necessary to obtain a complete protein monolayer was found to be less than 2 mol%. The adsorption isotherm of PNA to G_M1 was recorded and analyzed to be of Langmuir type, exhibiting a binding constant of PNA to the ganglioside of 8.3 ⋅ 10⁵ M^–1. The good agreement of the calculated Langmuir adsorption isotherm with the obtained experimental data implies that protein multilayers are not formed and that interactions between the adsorbents can be neglected. Furthermore, the association constants of two different saccharides, β-Galp-(1 → 3)-GalNAc exhibiting a strong binding to PNA in solution, and β-D-galactose with a much lower affinity were estimated by determining the equilibrium concentration of PNA attached to the surface. Moreover we were able to remove the attached lectin monolayer by digestion of the protein with pronase causing an increase in the resonant frequency which almost reversed the frequency shift to lower frequencies during adsorption. An even more complex system was built up by the use of digoxigenin-labeled PNA which also binds to the solid supported membrane containing the receptor G_M1. The immobilized lectin was recognized by anti-digoxigenin-F_ab-fragments, which is measurable by a further decrease of the resonant frequency. For all binding processes we found larger frequency shifts for a complete protein monolayer than predicted by Sauerbrey's equation, clearly showing that in addition to mass loading viscoelastic changes occur at the lipid-protein interface. Received: 22 July 1996 / Accepted: 12 September 1996     

Improvement of antibody immobilization using hyperbranched polymer and protein A

For the construction of a well-defined antibody surface, protein A was used as a binding material to immobilize antibodies onto gold-derivatized transducers. The traditional method tends to assemble protein A directly onto the gold-derivatized transducers. In this paper, we tried to indirectly bind protein A onto sensors through hyperbranched polymer (HBP) which was synthesized from p-phenylenediamine and trimesic acid. The three-dimensional structure of HBP and the characteristics including orientation control and biocompatibility of protein A led to highly efficient immunoreactions and enhanced detection system performance. With this strategy, cysteamine monolayer was first assembled onto Au electrodes associated with the piezoelectric quartz crystal; secondly, the cysteamine-modified gold electrode was further modified by the activated HBP; thirdly, protein A was immobilized onto the HBP film; and finally, antibodies were immobilized onto the surface of protein A film for detecting the corresponding antigen. The quartz crystal microbalance immunosensor thus fabricated was applied to detect hepatitis B surface antigen in solutions that ranged from 0.71 to 300 μg mL⁻¹. The detection limit was estimated to be 0.53 μg mL⁻¹. The immunosensor holds good selectivity, sensitivity, and repeatability.