Piezoelectric biosensors for biorecognition analysis: application to the kinetic study of HIV-1 Vif protein binding to recombinant antibodies

In this work three piezoelectric sensors modified with anti-HIV-1 Vif (virion infectivity factor) single fragment antibodies (4BL scFV), single domains (VH) and camelized single domains (VHD) were constructed and used to detect HIV1 Vif in liquid samples. Dithio-bis-succinimidyl-undecanoate (DSU) and 11-hydroxy-1-undecanethiol (HUT) mixed self assembled monolayers (SAM) were generated at the sensors surface onto which the antibodies were immobilized. All sensors detected specifically the target HIV1-Vif antigen in solution and no unspecific binding was monitored. Impedance analysis was performed to quantify electroacoustic and viscoelastic interferences during antibody immobilization and antigen recognition. The elimination of such interferences enabled the quantitative use of the piezoelectric immunosensors to estimate the antibody surface density as well as antigen binding and equilibrium constants. In spite of the possible limitation regarding mass transport and other related molecular phenomena, which were not considered in the binding model used, this work demonstrates the usefulness of piezoelectric biosensors in biorecognition analysis and evidences the advantages on using simultaneous impedance analysis to bring analytical significance to measured data, and thus to improve piezoelectric sensors sensitivity and applicability.     

An Electroacoustic Analysis for Determining the Effect of Amoxicillin on Microbial Cells

The probability of determining the effects of amoxicillin, which is one of β-lactam antibiotics, on microbial cells of Escherichia coli by the electroacoustic analysis method was shown for the first time. A piezoelectric resonator with a lateral electric field with a 1-mL liquid container was used as a biological sensor. It has been established that in the presence of amoxicillin the frequency dependence of the real and imaginary parts of the electrical impedance of a resonator loaded with a suspension of sensitive cells differs significantly from those of the resonator with a control of a microbial cell suspension without an antibiotic. When the resonator is loaded with the amoxicillin-resistant cell suspension, these dependencies are virtually the same. These results open prospects for the use of electroacoustic analysis methods to register the effect of β-lactam antibiotics on microbial cells and evaluate their antibacterial activity.     

Monitoring and kinetic parameter estimation for the binding process of berberine hydrochloride to bovine serum albumin with piezoelectric quartz crystal impedance analysis

A new method for monitoring, in real time, the drug-binding process to protein with piezoelectric quartz crystal impedance (PQCI) is proposed. The method was used to monitor the binding process of berberine hydrochloride to bovine serum albumin (BSA). BSA was immobilized on the silver electrode surface of a piezoelectric quartz crystal and the optimized experimental conditions were established. The BSA-coated piezoelectric sensor was in contact with berberine solution. The time courses of the resonant frequency and equivalent circuit parameters of the sensor during the protein-drug binding were simultaneously obtained. On the basis of the analysis of the multidimensional information provided by PQCI, it was concluded that the observed frequency decrease was mainly ascribed to the mass increase of the sensor surface resulting from the binding. According to the frequency decrease with time, the kinetics of the binding process were quantitatively studied. A piezoelectric response model for the binding was theoretically derived. Fitting the experimental data to the model, the kinetic parameters, such as the binding and dissociation rate constants (k(1) and k(-1)) and the binding equilibrium constant (K(a)), were determined. The k(1), k(-1), and K(a) values obtained at 25 degrees C were 67.5 (+/-0.1) (mol liter(-1))(-1) s(-1), 1.7 (+/- 0.1) x 10(-3) s(-1), and 3.97 (+/- 0.06) x10(4) (mol liter(-1))(-1), respectively.     

Micro-machined piezoelectric membrane-based immunosensor array

This paper reports a micro-machined piezoelectric membrane-based biosensor array for immunoassay. Goat immunoglobulin G (IgG) and HBsAg were immobilized as the probe molecules on the square piezoelectric membranes of the sensors that have dimensions of 3.5 microm x 500 microm x 500 microm. Due to the mass sensitive nature of these sensors, their resonant frequencies were depressed after the anti-goat IgG or anti-HBsAg was captured by the goat IgG or HBsAg. The resonant frequencies of the sensors were measured by an impedance analyzer. The experimental results demonstrate that the measured frequency change varies from 100 to 700 Hz, and the mass sensitivity of the device is estimated to be about 6.25 Hz/ng. A near linear relationship between the frequency change and the concentration of goat IgG was obtained, and the mass of the attached anti-goat IgG was calculated. The preliminary results discussed in this work indicate that the micro-machined piezoelectric membrane-based biosensor has a potential application as an immunosensor.     

Influence of electrolytes in the QCM response: discrimination and quantification of the interference to correct microgravimetric data

In this work we demonstrate that the presence of electrolytes in solution generates desorption-like transients when the resonance frequency is measured. Using impedance spectroscopy analysis and Butterworth-Van Dyke (BVD) equivalent electrical circuit modeling we demonstrate that non-Kanazawa responses are obtained in the presence of electrolytes mainly due to the formation of a diffuse electric double layer (DDL) at the sensor surface, which also causes a capacitor like signal. We extend the BVD equivalent circuit by including additional parallel capacitances in order to account for such capacitor like signal. Interfering signals from electrolytes and DDL perturbations were this way discriminated. We further quantified as 8.0+/-0.5 Hz pF-1 the influence of electrolytes to the sensor resonance frequency and we used this factor to correct the data obtained by frequency counting measurements. The applicability of this approach is demonstrated by the detection of oligonucleotide sequences. After applying the corrective factor to the frequency counting data, the mass contribution to the sensor signal yields identical values when estimated by impedance analysis and frequency counting.     

Quantitative Amyloid Imaging Using Image-Derived Arterial Input Function

Amyloid PET imaging is an indispensable tool widely used in the investigation, diagnosis and monitoring of Alzheimer’s disease (AD). Currently, a reference region based approach is used as the mainstream quantification technique for amyloid imaging. This approach assumes the reference region is amyloid free and has the same tracer influx and washout kinetics as the regions of interest. However, this assumption may not always be valid. The goal of this work is to evaluate an amyloid imaging quantification technique that uses arterial region of interest as the reference to avoid potential bias caused by specific binding in the reference region. 21 participants, age 58 and up, underwent Pittsburgh compound B (PiB) PET imaging and MR imaging including a time-of-flight (TOF) MR angiography (MRA) scan and a structural scan. FreeSurfer based regional analysis was performed to quantify PiB PET data. Arterial input function was estimated based on coregistered TOF MRA using a modeling based technique. Regional distribution volume (V_T) was calculated using Logan graphical analysis with estimated arterial input function. Kinetic modeling was also performed using the estimated arterial input function as a way to evaluate PiB binding (DVR_kinetic) without a reference region. As a comparison, Logan graphical analysis was also performed with cerebellar cortex as reference to obtain DVR_REF. Excellent agreement was observed between the two distribution volume ratio measurements (r>0.89, ICC>0.80). The estimated cerebellum V_T was in line with literature reported values and the variability of cerebellum V_T in the control group was comparable to reported variability using arterial sampling data. This study suggests that image-based arterial input function is a viable approach to quantify amyloid imaging data, without the need of arterial sampling or a reference region. This technique can be a valuable tool for amyloid imaging, particularly in population where reference normalization may not be accurate.     

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.     

Immunodetection of bacteriophages by a piezoelectric resonator with lateral electric field

It has been demonstrated that electroacoustic analysis with polyclonal antibodies can be used for bacteriophage detection. The frequency dependences of the real and imaginary parts of electrical impedance of a resonator with a viral suspension with antibodies were shown to be essentially different from the dependences of a resonator with control viral suspension without antibodies. It was shown that ΦAl-Sp59b bacteriophages were detected with the use of antibodies in the presence of foreign virus particles. The ΦAl-Sp59b bacteriophage content in the analyzed suspension was ~10¹⁰–10⁶ phages/mL; the time of analysis was no more than 5 min. The optimally informative parameter for obtaining reliable information was the change in the real or imaginary part of electrical impedance at a fixed frequency near the resonance upon the addition of specific antibodies to the analyzed suspension. It was demonstrated that the interaction between bacteriophages and antibodies can be recorded, offering good prospects for the development of a biological sensor for liquid-phase identification and virus detection.     

Affinity-selected filamentous bacteriophage as a probe for acoustic wave biodetectors of Salmonella typhimurium

Proof-in-concept biosensors were prepared for the rapid detection of Salmonella typhimurium in solution, based on affinity-selected filamentous phage prepared as probes physically adsorbed to piezoelectric transducers. Quantitative deposition studies indicated that approximately 3 x 10(10)phage particles/cm(2) could be irreversibly adsorbed for 1 h at room temperature to prepare working biosensors. The quality of phage deposition was monitored by fluorescent microscopy. Specific-bacterial binding resulted in resonance frequency changes of prepared sensors, which were evaluated using linear regression analysis. Sensors possessed a rapid response time of <180 s, had a low-detection limit of 10(2)cells/ml and were linear over a range of 10(1)-10(7)cells/ml with a sensitivity of 10.9 Hz per order of magnitude of S. typhimurium concentration. Viscosity effects due to increasing bacterial concentration and non-specific binding were not significant to the piezoelectric platform as confirmed by dose-response analysis. Phage-bacterial binding was confirmed by fluorescence and scanning electron microscopy. Overall, phage may constitute effective bioreceptors for use with analytical platforms for detecting and monitoring bacterial agents, including use in food products and possibly biological warfare applications.     

Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance

A biosensor based on bovine serum albumin (BSA) for the detection of lead (Pb(2+)) ion was developed and characterized. BSA was immobilized onto a colloidal Au-modified piezoelectric quartz crystal (PQC) as a biosensor for the detection of Pb(2+) ion by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb(2+) ion showed excellent linearity throughout the concentration range from 1.0 x 10(-7) to 3.0 x 10(-9)mol/L. The interaction between the Pb(2+) ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 x 10(-9)mol/L for Pb(2+) was obtained. Kinetic parameters of the Pb(2+)-BSA interactions were also determined by using this chip. The sensor chip could be regenerated for use by dipping in the ethylenediaminetetraacetic acid (EDTA) solution for approximately 2h, and the chip was used to detect Pb(2+) ion for eight times without obvious signal attenuation.     

Dual-polarization interferometry: an analytical technique to measure changes in protein structure in real time, to determine the stoichiometry of binding events, and to differentiate between specific and nonspecific interactions

The study of solution-phase interactions between small molecules and immobilized proteins is of intense interest, especially to the pharmaceutical industry. An optical sensing technique, dual polarization interferometry, has been employed for the detailed study of a model protein system, namely, d-biotin interactions with streptavidin immobilized on a solid surface. Changes in thickness and density of an immobilized streptavidin layer as a result of the binding of d-biotin have been directly measured in solution and in real time. The results obtained from this approach are in excellent agreement with X-ray crystallographic data for the structural changes expected in the streptavidin-D-biotin system. The mass changes measured on binding d-biotin also agree closely with anticipated binding capacity values. Determination of the density changes occurring in the protein adlayer provides a means for differentiation between specific and nonspecific interactions.     

Streptavidin contains an RYD sequence which mimics the RGD receptor domain of fibronectin

Streptavidin binds at low levels and high affinity to cell surfaces, the cause of which can be traced to the occurrence of a sequence containing RYD (Arg-Tyr-Asp) in the protein molecule. This binding is enhanced in the presence of biotin. Cell-bound streptavidin can be displaced by fibronectin, as well as by RGD- and RYD-containing peptides. In addition, streptavidin can displace fibronectin from cell surfaces. The RYD sequence of streptavidin thus mimics RGD (Arg-Gly-Asp), the universal recognition domain present in fibronectin and other adhesion-related molecules. The observed adhesion to cells has no relevance to biotin-binding since the RYD sequence is not part of the biotin-binding site of streptavidin. Since the use of streptavidin in avidin-biotin technology is based on its biotin-binding properties, researchers are hereby warned against its indiscriminate use in histochemical and cytochemical studies.     

Methods for measuring acoustic power of an ultrasonic neurosurgical device

Measurement of the acoustic power in high-energy ultrasonic devices is complex due to occurrence of the strong cavitation in front of the sonotrode tip. In our research we used three methods for characterization of our new ultrasonic probe for neuroendoscopic procedures. The first method is based on the electromechanical characterization of the device measuring the displacement of the sonotrode tip and input electrical impedance around excitation frequency with different amounts of the applied electrical power The second method is based on measuring the spatial pressure magnitude distribution of an ultrasound surgical device produced in an anechoic tank. The acoustic reciprocity principle is used to determinate the derived acoustic power of equivalent ultrasound sources at frequency components present in the spectrum of radiated ultrasonic waves. The third method is based on measuring the total absorbed acoustic power in the restricted volume of water using the calorimetric method. In the electromechanical characterization, calculated electroacoustic efficiency factor from equivalent electrical circuits is between 40-60%, the same as one obtained measuring the derived acoustic power in an anechoic tank when there is no cavitation. When cavitation activity is present in the front of the sonotrode tip the bubble cloud has a significant influence on the derived acoustic power and decreases electroacoustic efficiency. The measured output acoustic power using calorimetric method is greater then derived acoustic power, due to a large amount of heat energy released in the cavitation process.     

Instrumental noise estimates stabilize and quantify endothelial cell micro-impedance barrier function parameter estimates

A novel transcellular micro-impedance biosensor, referred to as the electric cell-substrate impedance sensor or ECIS, has become increasingly applied to the study and quantification of endothelial cell physiology. In principle, frequency dependent impedance measurements obtained from this sensor can be used to estimate the cell–cell and cell–matrix impedance components of endothelial cell barrier function based on simple geometric models. Few studies, however, have examined the numerical optimization of these barrier function parameters and established their error bounds. This study, therefore, illustrates the implementation of a multi-response Levenberg–Marquardt algorithm that includes instrumental noise estimates and applies it to frequency dependent porcine pulmonary artery endothelial cell impedance measurements. The stability of cell–cell, cell–matrix and membrane impedance parameter estimates based on this approach is carefully examined, and several forms of parameter instability and refinement illustrated. Including frequency dependent noise variance estimates in the numerical optimization reduced the parameter value dependence on the frequency range of measured impedances. The increased stability provided by a multi-response non-linear fit over one-dimensional algorithms indicated that both real and imaginary data should be used in the parameter optimization. Error estimates based on single fits and Monte Carlo simulations showed that the model barrier parameters were often highly correlated with each other. Independently resolving the different parameters can, therefore, present a challenge to the experimentalist and demand the use of non-linear multivariate statistical methods when comparing different sets of parameters.     

An automated analysis of highly complex flow cytometry-based proteomic data

The combination of color-coded microspheres as carriers and flow cytometry as a detection platform provides new opportunities for multiplexed measurement of biomolecules. Here, we developed a software tool capable of automated gating of color-coded microspheres, automatic extraction of statistics from all subsets and validation, normalization, and cross-sample analysis. The approach presented in this article enabled us to harness the power of high-content cellular proteomics. In size exclusion chromatography-resolved microsphere-based affinity proteomics (Size-MAP), antibody-coupled microspheres are used to measure biotinylated proteins that have been separated by size exclusion chromatography. The captured proteins are labeled with streptavidin phycoerythrin and detected by multicolor flow cytometry. When the results from multiple size exclusion chromatography fractions are combined, binding is detected as discrete reactivity peaks (entities). The information obtained might be approximated to a multiplexed western blot. We used a microsphere set with >1,000 subsets, presenting an approach to extract biologically relevant information. The R-project environment was used to sequentially recognize subsets in two-dimensional space and gate them. The aim was to extract the median streptavidin phycoerythrin fluorescence intensity for all 1,000+ microsphere subsets from a series of 96 measured samples. The resulting text files were subjected to algorithms that identified entities across the 24 fractions. Thus, the original 24 data points for each antibody were compressed to 1-4 integrated values representing the areas of individual antibody reactivity peaks. Finally, we provide experimental data on cellular protein changes induced by treatment of leukemia cells with imatinib mesylate. The approach presented here exemplifies how large-scale flow cytometry data analysis can be efficiently processed to employ flow cytometry as a high-content proteomics method.     

Study on the interaction between DNA and protein induced by anticancer drug carboplatin

The interaction of DNA and human serum albumin (HSA) in the presence of anticancer drug carboplatin was studied with piezoelectric quartz crystal impedance (PQCI) and electrochemistry techniques. In the PQCI analysis, the correlative parameters including the frequency (f0), the motional resistance (R(m)), and the static capacitance (C0) in the experiment were obtained and discussed in detail. Additionally, the kinetics parameters of the cross-linking process were investigated and a response kinetics model was deduced. The values of association rate constant k1, dissociation rate constant k(-1) and the reaction equilibrium constant K were estimated to be 1.895 mg/ml(-1) s(-1), 4.7 x 10(-5) s(-1) and 4.03 x 10(4) (mg/ml)-1, respectively. Furthermore, cyclic voltammetry (CV) and electrochemical AC impedance techniques were employed to testify the cross-linking process.     

Dual polarization interferometry characterization of carbohydrate-protein interactions

Dual polarization interferometry (DPI) is an analytical technique that allows the simultaneous determination of thickness, density, and mass of a biological layer on a sensing waveguide surface in real time. The technique was applied to the analysis of carbohydrate-protein interactions. The selected system involved a 12-kDa recombinant fragment of collagen V (HepV) and heparin, a complex polysaccharide. Here we report on the analysis of thickness, density, and mass of surface structures obtained during the binding of HepV to heparin, which is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. This system, which was initially studied for its biological relevance, displayed anomalous behavior in kinetic studies using surface plasmon resonance (SPR) assays that has been attributed to putative conformational changes. It was this putative conformational change that prompted us to investigate the binding using an alternative analytical approach. While using DPI to monitor binding events, a streptavidin layer (surface coverage 2.105 ng mm(-2)) was bound to the sensor surface (92% coverage), which captured 0.105 ng mm(-2) of biotinylated heparin (a stoichiometric ratio of 1:6 heparin-streptavidin). The heparin inserted into the streptavidin layer but was still found to be capable of binding 0.154 ng mm(-2) of HepV, which was also observed to insert into the streptavidin layer. This allowed the reliable calculation of the stoichiometric ratio for the HepV-heparin complex ( approximately 1.7:1.0), which has proved to be difficult to evaluate by SPR assays. Furthermore, real-time analysis of the heparin-HepV interaction by DPI suggested that there was some surface loss (probably of streptavidin) while the binding was occurring rather than the putative conformational change that has been suggested on the basis of kinetic data alone. This gives further insight into the binding mechanism of HepV to heparin.     

Molecularly imprinted thin film self-assembled on piezoelectric quartz crystal surface by the sol-gel process for protein recognition

A novel method of combining sol-gel and self-assembly technology to prepare a human serum albumin (HSA)-imprinted film on the surface of piezoelectric quartz crystal (PQC) Au-electrode modified with thioglycolic acid was described in this paper. The imprinting process was characterized by using the piezoelectric quartz crystal impedance (PQCI) technique and electrochemical impedance technique. Scanning electron microscope (SEM) was employed to characterize the surface morphology of the resultant imprinted film. The piezoelectric technique and electrochemical impedance technique were also employed to investigate the binding performance of the sol-gel-imprinted film with the template protein. The results showed that the imprinted PQC film can give selective recognition to the template protein. The effects of salts and solvents on the binding capacity of the imprinted film with protein were discussed in detail. Other influencing factors (temperature and pH) have also been investigated. This self-assembly sol-gel imprinting technique was proved to be an alternative method for the preparation of biomacromolecule-imprinted thin film.     

Study of erythrocyte sedimentation behavior by piezoelectric crystal impedance sensor

Based on the impedance characteristic of erythrocytes at high frequency, the response of piezoelectric crystal impedance (PCI) sensor in the erythrocyte suspension was derived and verified experimentally. A method of using PCI sensor to investigate erythrocyte aggregation-sedimentation phenomenon was proposed. From the frequency response of the PCI sensor, the erythrocyte aggregation time and sedimentation rate could be obtained during erythrocyte aggregation and sedimentation. With the present method, the effects of the erythrocyte deformability, the osmotic pressure and the coexisting macromolecules on the erythrocyte sedimentation rate were studied. The results show that the PCI sensor possesses some advantages, such as good sensitivity, simplicity of use and no thermal effect for the impedance study of erythrocyte aggregation and sedimentation.