Thomas M.A. Gronewold

SAW Sensor System for Marker‐Free Molecular Interaction Analysis

Abstract A chip‐based biosensor system for marker‐free detection of affinity and molecular interaction kinetics was developed. It applies surface‐acoustic‐waves. Combined with a novel double‐frequency measurement‐mode, which assures reliable reference measurements, it allows a precise real‐time detection of mass loading and viscosity alteration. The system is equipped with five independently working channels, which enable it to simultaneously perform different, e.g., reference measurements. Exemplarily performed investigations of the association of thrombin and antithrombin antibodies demonstrated the applicability of the system to affinity and kinetic analyses. The system yielded a mean value for the equilibrium dissociation constant of K D=116±9 nM, which compares well with the supplier specification.

Surface Acoustic Wave Biosensor as a Tool to Study the Interaction of Antimicrobial Peptides with Phospholipid and Lipopolysaccharide Model Membranes

Surface acoustic wave biosensors are a powerful tool for the study of biomolecular interactions. The modulation of a surface-confined acoustic wave is utilized here for the analysis of surface binding. Phase and amplitude of the wave correspond roughly to mass loading and viscoelastic properties of the surface, respectively. We established a procedure to reconstitute phospholipid and lipopolysaccharide bilayers on the surface of a modified gold sensor chip to study the mode of action of membrane-active peptides. The procedure included the formation of a self-assembled monolayer of 11-mercaptoundecanol, covalent coupling of carboxymethyl-dextran, and subsequent coating with a poly- l-lysine layer. The lipid coverage of the surface is highly reproducible and homogeneous as demonstrated in atomic force micrographs. Ethanol/triton treatment removed the lipids completely, which provided the basis for continuous sequences of independent experiments. The setup was applied to investigate the binding of human cathelicidin-derived peptide LL32, as an example for antimicrobial peptides, to immobilized phosphatidylserine membranes. The peptide-membrane interaction results in a positive phase shift and an increase in amplitude, indicating a mass increase along with a loss in viscosity. This suggests that the bilayer becomes more rigid upon interaction with LL32.

Microfabricated high-performance microwave impedance biosensors for detection of aptamer-protein interactions

High-frequency impedance biosensors with nanometer gaps have been prepared for the detection of biomolecular interactions such as protein-antibody and protein-aptamer binding. The sensor principle is based on electrical impedance changes measured at 1.2 GHz due to changes of the effective dielectric constant within the 68 nm gaps between two gold electrodes. As a model system, the specific binding of the blood clotting factor human thrombin with different concentrations to its ribonucleic acid (RNA) α-thrombin aptamer, as well as the immobilization process of the RNA-aptamer, have been detected in real time. By using a similar 68 nm-gap sensor blocked with bovine serum albumin and a reference sensor with 10μm electrode spacing, signal changes due to variations of the bulk dielectric constant due to buffer/analyte solutions, and unspecific binding events have been analyzed.

Kinetic Binding Analysis of Aptamers Targeting HIV-1 Proteins by a Combination of a Microbalance Array and Mass Spectrometry (MAMS)

An enhanced chip-based detection platform was developed by integrating a surface acoustic wave biosensor of the Love-wave type with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). The system was applied to characterize the interaction of aptamers with their cognate HIV-1 proteins. The aptamers, which target two proteins of HIV-1, were identified using an automated in vitro selection platform. For aptamers S66A-C6 and S68B-C5, which target the V3 loop of the HIV-1 envelope protein gp120, KD values of 406 and 791 nM, respectively, were measured. Aptamer S69A-C15 was shown to bind HIV-1 reverse transcriptase (HIV-1 RT) with a KD value of 637 nM when immobilized on the biosensor surface. HIV-1 RT was identified with high significance using MALDI-ToF MS even in crude protein mixtures. The V3-loop of gp120 could be directly identified when using chip-bound purified protein samples. From crude protein mixtures, it could be identified indirectly with high significance via its fusion-partner glutathione-S-transferase (GST). Our data show that the combination of the selectivity of aptamers with a sensitive detection by MS enables the reliable and quantitative analysis of kinetic binding events of protein solutions in real time.

Combination of a SAW-biosensor with MALDI mass spectrometric analysis

A S-sens K5 surface acoustic wave biosensor was coupled with mass spectrometry (SAW-MS) for the analysis of a protein complex consisting of human blood clotting cascade factor alpha-thrombin and human antithrombin III, a specific blood plasma inhibitor of thrombin. Specific binding of antithrombin III to thrombin was recorded as a function of time with a S-sens K5 biosensor. Two out of five elements of the sensor chip were used as references. To the remaining three elements coated with RNA anti-thrombin aptamers, thrombin and antithrombin III were bound consecutively. The biosensor measures mass changes on the chip surface showing that 20% of about 400fmol/cm2 thrombin formed a complex with the 1.7-times larger antithrombin III. Mass spectrometry (MS) was applied to identify the bound proteins. Sensor chips with aptamer-captured (1) thrombin and (2) thrombin-antithrombin III complex (TAT-complex) were digested with proteases on the sensor element and subsequently identified by peptide mass fingerprint (PMF) with matrix assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry. A significant identification of thrombin was achieved by measuring the entire digest with MALDI-ToF MS directly from the sensor chip surface. For the significant identification of both proteins in the TAT-complex, the proteolytic peptides had to be separated by nano-capillary-HPLC prior to MALDI-ToF MS. SAW-MS is applicable to protein interaction analysis as in functional proteomics and to miniaturized diagnostics.

Selection process generating peptide aptamers and analysis of their binding to the TiO2 surface of a surface acoustic wave sensor.

The set-up presented in this article is intended for the selection of peptides which serve as specific binders to suitable materials. Additionally, the interaction of such binders with material surfaces can be characterized. Using this approach, a subset of peptides which adhere to the mineral TiO(2) was generated by means of a cell surface display library. The peptides are constrained by a thioredoxin scaffold. Selection of proteins was carried out on a silicium wafer sputtered with TiO(2) in anatase conformation. To verify binders and to analyze the binding kinetics of the diluted suspension of the purified proteins, the chip-based S-sens K5 surface acoustic wave sensor system was used. The surface of the sensor chips was also TiO(2), resembling the material of the Si wafer selection target retaining the peptides. Several peptides were identified. The respective binding behavior differed. The data derived from real-time interaction analysis were evaluated to select for strong and specific binders. For one of these peptides, the binding kinetics was analyzed. On- and off-rate binding constants were extracted from the fitted curves. With the resulting association rate constant k(on) and the dissociation constant k(off), the affinity of the peptide for the TiO(2) surface was calculated, represented by the equilibrium dissociation constant K(D)=81 nM.

Lithium Tantalate Surface Acoustic Wave Sensors for Bio-Analytical Applications

A novel chip-based measurement system for marker-free detection of affinity and molecular interaction kinetics has been developed. Lithium tantalate (LiTaO3) based Love-wave sensors are utilized as an alternative to quartz as substrate material in order to improve both the sensitivity and the limit-of-detection (LOD). Monitoring the coupling of thrombin to its antibody at low concentrations 1 nM and 10 nM showed phase changes of Deltaphi = 0.10deg and 0.35deg, respectively, for an active sensor area of 5.6 mm2. We have found that the estimated detection limit for thrombin is on the order of 0.18 nM for our LiTaO3 sensors.

Measurement of porcine haptoglobin in meat juice using surface acoustic wave biosensor technology

Aim of the study was the application of biosensor technique to measure the concentration of an acute phase protein (APP) within complex matrices from animal origin. For the first time, acute phase protein haptoglobin (Hp) was detected from unpurified meat juice of slaughter pigs by a label-free biosensor-system, the SAW-based sam®5 system. The system uses a sensor chip with specific antibodies to catch Hp while the mass-related phase shift is measured. The concentration is calculated as a function of these measured phase shifts. The results correlate very well with reference measurement results obtained by enzyme-linked immunosorbent assay (ELISA), R=0.98. The robust setup of the surface acoustic wave (SAW)-based system and its ability to measure within very short time periods qualifies it for large-scale analyses and is apt to identify rapidly pigs in the meat production process whose consumption would have an increased risk for consumers.

Surface Acoustic Biosensors using Aptamers as Ligands

A Love-wave biosensor array has been designed by coupling aptamers to the surface of a Love-wave sensor chip. The sensor chip consists of five sensor elements and allows label-free, real-time, and quantitative measurements of protein binding events in concentration-dependent fashion. The biosensor was calibrated for human α-thrombin and HIV-1 Rev peptide by binding fluorescence labelled molecules and correlating the mass of the bound molecules to fluorescence intensity. Detection limits of approximately 75 pg/cm2 were obtained, and analyte recognition was shown to be specific. The sensor may easily be regenerated by simple washing steps.