Development of a parallelizable QCM-D array for the mass spectrometric analysis of proteins

Quarz crystal microbalances allow the monitoring of the adsorption process of mass from a liquid to their surface. The adsorbed mass can be analysed regarding to its protein content using mass spectromety. To ensure the protein identification the results of several measurements can be combined. A high content QCM-D array was developed to allow up to ten measurements parallel. The samples can be routed inside the array distributing one sample to several chips. The fluidic parts were prototyped using 3D printing. The assembled array was tight and the sample routing function could be demonstrated. A temperature controller was developed and implemented. The parameters for the PID controller were determined and the controller was shown to be able to keep the temperature constant over long time with high accuracy

Melanocortin-4 receptor gene: case-control study and transmission disequilibrium test confirm that functionally relevant mutations are compatible with a major gene effect for extreme obesity

We initially performed a mutation screen of the coding region of the MC4R in 808 extremely obese children and adolescents and 327 underweight or normal-weight controls allowing for a case-control study. A total of 16 different missense, nonsense, and frameshift mutations were found in the obese study group; five of these have not been observed previously. In vitro assays revealed that nine [the haplotype (Y35X; D37V) was counted as one mutation] of the 16 mutations led to impaired cAMP responses, compared with wild-type receptor constructs. In contrast, only one novel missense mutation was detected in the controls, which did not alter receptor function. The association test based on functionally relevant mutations was positive (P = 0.006, Fisher's exact test, one-sided). We proceeded by screening a total of 1040 parents of 520 of the aforementioned obese young index patients to perform transmission disequilibrium tests. The 11 parental carriers of functionally relevant mutations transmitted the mutation in 81.8% (P = 0.033; exact one-sided McNemar test). These results support the hypothesis that these MC4R mutations represent major gene effects for obesity

Surface Acoustic Wave (SAW) Resonators for Monitoring Conditioning Film Formation

We propose surface acoustic wave (SAW) resonators as a complementary tool for conditioning film monitoring. Conditioning films are formed by adsorption of inorganic and organic substances on a substrate the moment this substrate comes into contact with a liquid phase. In the case of implant insertion, for instance, initial protein adsorption is required to start wound healing, but it will also trigger immune reactions leading to inflammatory responses. The control of the initial protein adsorption would allow to promote the healing process and to suppress adverse immune reactions. Methods to investigate these adsorption processes are available, but it remains difficult to translate measurement results into actual protein binding events. Biosensor transducers allow user-friendly investigation of protein adsorption on different surfaces. The combination of several transduction principles leads to complementary results, allowing a more comprehensive characterization of the adsorbing layer. We introduce SAW resonators as a novel complementary tool for time-resolved conditioning film monitoring. SAW resonators were coated with polymers. The adsorption of the plasma proteins human serum albumin (HSA) and fibrinogen onto the polymer-coated surfaces were monitored. Frequency results were compared with quartz crystal microbalance (QCM) sensor measurements, which confirmed the suitability of the SAW resonators for this application

Surface Acoustic Wave (SAW) Biosensors to observe the formation of conditioning films

Biofilme können in technischen Systemen zu Beschädigungen oder Beeinträchtigungen der Leistung des Systems führen. In der Humanmedizin können sie zu Infektionen an Kathetern und Implantat-oberflächen führen. Biofilme bilden sich an Phasengrenzflächen. Der erste Schritt in der Entstehung eines Biofilms ist die Bildung eines "Conditioning"-Films aus Substanzen des umgebenden Mediums. Ein zur Beschichtung von Implantaten eingesetztes Polymer ist Parylen C. Auf akustischen Oberflächenwellen, englisch: surface acoustic wave (SAW), basierende Sensoren zeigen bei Änderungen der Eigenschaften ihrer aktiven Oberfläche eine Änderung ihrer Resonanzfrequenz. Im Rahmen dieser Arbeit wurde überprüft, ob sich SAW-Sensoren zur Echtzeitbeobachtung der Bildung von "Conditioning"-Filmen eignen. Als Vergleichssystem wurden Schwingquarze, englisch: quarz crystal microbalances (QCM), gewählt, da diese ebenfalls gravimetrischen Sensoren bereits zur Beoabachtung von "Conditioning"-Filmen eingesetzt werden. Sensoren beider Systeme wurden mit Parylen C beschichtet und jeweils mit drei relevanten Proteinen des menschlichen Blutplasmas beprobt. Es konnte gezeigt werden, dass sich auf akustischen Oberflächenwellen basierende Sensoren zur Beobachtug der Adsorption von Plasmaproteinen auf Parylen C eignen. Zudem konnten auf SAW-Sensoren reproduzierbar Charakteristiken in den Kurvenverläufen beobachtet werden, welche beim Vergleichssystems nicht sichtbar waren.Biofilms can destroy parts of technical systems or decrease their efficiency. Infections at the surfaces of medical devices, like catheters or implants, can also result from biofilm formation. Biofilms occur at solid-liquid interfaces. The first step in the formation formation of a biofilm is the formation of a conditioning film by compounds of the liquid phase. Parylene C is a polymer that is often used as a biocompatible coating for implants. The resonance frequency of surface acoustic wave (SAW) sensors changes, if the conditions at their active surfaces change. The topic of this thesis was to show, if SAW-sensors can be used to observe the formation of a conditioning film in real time. As reference system quarz crystal microbalances (QCM) were selected, because these sensors were used to observe conditioning film formation before. Sensors of both systems were used to observe the adsorption of three proteins, that are part of human plasma, on parylene C coated surfaces. It could be shown, that surface acoustic wave sensors can be used to observe the formation of conditioning films on parylene C in real time. Furthermore, SAW-sensors allowed the reproducible observation of signal characteristics, which could not be observed with the QCM-sensors

A new data processing routine facilitating the identification of surface adhered proteins from bacterial conditioning films via QCM-D/MALDI-ToF/MS

Conditioning films are an important factor in the initiation and development of microbial biofilms, which are the leading cause of chronic infections associated with medical devices. Here, we analyzed the protein content of conditioning films formed after exposure to supernatants of cultures of the human pathogen Pseudomonas aeruginosa PAO1. Adhesion of substances from the supernatant was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) sensor chips modified with the commonly used implant material titanium dioxide (TiO2). Attached proteins were identified after on-chip digestion using matrix-assisted laser desorption/ionization (MALDI) time of flight (ToF) mass spectrometry (MS), and a new data processing tool consisting of an XML-database with theoretical tryptic peptides of every PAO1 protein and PHP scripts. Sub-databases containing only proteins, that we found in all replicates, were created and used for MS/MS precursor selection. The obtained MS/MS peaklists were then matched against theoretical fragmentations of the expected peptide sequences to verify protein identification. Using this approach we were able to identify 40 surface-associated proteins. In addition to extracellular proteins such as adhesins, a number of intra-cellular proteins were identified which may be involved in conditioning film formation, suggesting an as-yet unidentified role for these proteins, possibly after cell lysis. [Figure not available: see fulltext.