Sudden aggravations of chronic inflammatory airway diseases are difficult-to-foresee life-threatening episodes for which advanced prognosis-systems are highly desirable. Here we present an experimental chip-based fluidic system designed for the rapid and sensitive measurement of biomarkers prognostic for potentially imminent asthma or COPD exacerbations. As model biomarkers we chose three cytokines (interleukin-6, interleukin-8, tumor necrosis factor alpha), the bacterial infection marker C-reactive protein and the bacterial pathogen Streptococcus pneumoniae-all relevant factors in exacerbation episodes. Assay protocols established in laboratory environments were adapted to 3D-printed fluidic devices with emphasis on short processing times, low reagent consumption and a low limit of detection in order to enable the fluidic system to be used in point-of-care settings. The final device demonstrator was validated with patient sample material for its capability to detect endogenous as well as exogenous biomarkers in parallel.

Crossing cellular membranes is a versatile molecular property that allows for a wide variety of peptides with cell penetrating capabilities. This broadness complicates identification of candidates suited best for a specific application. To facilitate the screening of this enormous molecular space in a supervised manner we here present a method to "breed" the desired molecules by applying the rules of Darwinian evolution. With this mate-and-check protocol, which combines an in silico evolution step with an in vitro performance test, cell penetrating peptides that are optimized for a specific task can be achieved in a few rounds of breeding. The procedure is simple and straightforward on the synthetic site but requires robust, highly reproducible and close-to-reality biological assays to yield realistic functional output. With this technology even top-performing peptides can be further improved and functionally adjusted.

All solid-phase immunoassay techniques depend on so-called blocking reagents to suppress the background that is caused by unwanted adhesion of assay system components to the solid support. Commonly used blocking reagents based on biological materials bear severe inherent drawbacks such as heterogeneity and cross-reactivity, while synthetic alternatives often show insufficient background prevention. In this study, polyethylene glycol-conjugated alkylamines were synthesized via a versatile building block approach and were studied as novel blocking reagent candidates in immunoassays. The newly developed substances outperformed commonly used blocking reagents in two different ELISA setups, enabling both, excellent prevention of non-specific binding and particularly high assay sensitivity. This class of surfactants therefore may contribute significantly to the field of assay technology.

Nanoscience has flourished with increasing use of nanoparticles in many products. The particles enter the environment and affect both biotic and abiotic components of the ecosystem. Via the water supply and the food chain, humans could be affected by ingesting those particles. In this chapter, we will discuss mechanisms by which nanoparticles or their constituents can be translocated from the gastrointestinal tract, what their fate may be and how relevant this is for human health.

Delivering reagents into cells is a key demand in molecular medicine. The vehicle of choice is often cell penetrating peptides (CPPs), which can ferry conjugated cargo across membranes. Although numerous peptides have been shown to promote such uptake events, there has been no comprehensive comparison of individual performance under standardized conditions. We have devised a method to rapidly analyze the ability of a multitude of CPP conjugates to carry a model cargo into HeLa cells. Sequence information for 474 CPPs was collected from literature sources, and the respective peptides were synthesized and modified with carboxyfluorescein (FAM) as model cargo. All candidates were evaluated in an identical uptake test, and transport was quantified using cellular fluorescence intensities. Substantial differences in the ability to carry the fluorophore into the cells were observed, with transport performance differing by a factor of 70 between the best CPP investigated and cargo alone. Strong correlations were observed between uptake efficiency and both sequence length and the presence of positive net charge. A compilation of the 20 top performers with regard to cargo delivery performance and cell compatibility is provided.

In nature, building block-based biopolymers can adapt to functional and environmental demands by recombination and mutation of the monomer sequence. We present here an analogous, artificial evolutionary optimization process which we have applied to improve the functionality of cell-penetrating peptide molecules. The "evolution" consisted of repeated rounds of in silico peptide sequence alterations using a genetic algorithm followed by in vitro peptide synthesis, experimental analysis, and ranking according to their "fitness" (i.e., their ability to carry the cargo carboxyfluorescein into cultured cells). The genetic algorithm-based optimization method was customized and adapted from former successful applications in the lab to realize an early convergence and a minimum number of in vitro and in silico processing steps by configured settings derived from empirical in silico simulation. We started out with 20 "lead peptides" which we had previously identified as top performers regarding their ability to enter cultured cells. Ten breeding rounds comprising 240 peptides each yielded a peptide population of which the top 10 candidates displayed a 6-fold (median values) increase in its cell-penetration capability compared with the top 10 lead peptides, and two consensus sequences emerged which represent local fitness optima. In addition, the cell-penetrating potential could be proven independently of the carboxyfluorescein cargo in an alternative setting. Our results demonstrate that we have established a powerful optimization technology that can be used to further improve peptides with known functionality and adapt them to specific applications.

This paper presents a CMOS radio frequency dielectric sensor platform for the detection of relative viscosity changes in sputum-mucin. The operating frequency of the sensor is 12 GHz. The sensor chip has been fabricated in 250 nm SiGe:C BiCMOS technology of IHP. The work shows that the sensor can distinguish between a highly viscous clot of sputum-mucin and dilute sputum-mucin. The viscosity of sputum is often regarded as a distinguishing feature of lung diseases and therefore, the sensor's capability of rapid detection of sputum viscosity makes it suitable for fast screening of lung diseases. A change of 200 MHz in the resonant frequency is shown for a change of 60% in the sputum-mucin viscosity. The sensor shown in this work is first of its kind to be used for detecting viscosity changes in a biological sample and is proposed to be used for future medical diagnostic tool for rapid screening of infectious lung diseases.

In chronic inflammatory airway diseases, mucins display disease-related alterations in quantity, composition and glycosylation. This opens the possibility to diagnose and monitor inflammatory airway disorders and their exacerbation based on mucin properties. For such an approach to be reasonably versatile and diagnostically meaningful, the mucin of interest must be captured in a reliable, patient-independent way. To identify appropriate mucin-specific reagents, we tested anti-mucin antibodies on mucin-content-standardized, human bronchoalveolar lavage fluid samples in immunoblot assays. All commercially available monoclonal antibodies against the major airway mucin MUC5AC were screened, except for those with known specificity for carbohydrates, as glycosylation patterns are not mucin-specific. Our results indicated considerable inter-patient and inter-antibody variability in mucin recognition for all antibodies and samples tested. The best results in terms of signal strength and reproducibility were obtained with antibodies Mg-31, O.N.457 and 45M1. Additional epitope mapping experiments revealed that only one of the antibodies with superior binding to MUC5AC recognized linear peptide epitopes on the protein backbone.

Detection of cancer at an early stage is pivotal for successful treatment and long term survival, yet early diagnosis requires sensitive and specific markers that can be easily detected by screening procedures. Differences in the surface structure of tumor and healthy cells, if sufficiently pronounced and discernible, may serve that purpose. We analyzed the luminal surface of healthy and neoplastic human colorectal tissues for the presence and architecture of the glycocalyx a dense network of highly glycosylated proteins using transmission electron microscopy. The ultrastructural analyses showed that 93% of healthy mucosae were covered by an intact glycocalyx. Contrarily, on over 90% of the surface of neoplastic cells the glycocalyx was absent. The sensitivity and specificity of our marker "absence of a glycocalyx" are excellent, being 91% (83-96%) and 96% (89-99%) for adenocarcinomas and 94% (73-100%) and 92% (85-97%) for precancerous polyps (means and 95% confidence intervals). Using a cell culture model we could demonstrate that a particulate probe targeting a cell surface receptor usually concealed beneath the glycocalyx can bind selectively to glycocalyx-free areas of a tumor cell layer. We propose that the absence of a glycocalyx may serve as novel type of tumor marker. If the absence of the glycocalyx can be detected e.g. via binding of imaging probes to non-shielded surface receptors of anomalously differentiated cells, this tumor marker could be used to enable early diagnosis of colorectal cancer.

Background: Anthropogenic nanoparticles (NPs) have found their way into many goods of everyday life. Inhalation, ingestion and skin contact are potential routes for NPs to enter the body. In particular the digestive tract with its huge absorptive surface area provides a prime gateway for NP uptake. Considering that NPs are covered by luminal gut-constituents en route through the gastrointestinal tract, we wanted to know if such modifications have an influence on the interaction between NPs and enterocytes. Results: We investigated the consequences of a treatment with various luminal gut-constituents on the adherence of nanoparticles to intestinal epithelial cells. Carboxylated polystyrene particles 20, 100 and 200 nm in size represented our anthropogenic NPs, and differentiated Caco-2 cells served as model for mature enterocytes of the small intestine. Pretreatment with the proteins BSA and casein consistently reduced the adherence of all NPs to the cultured enterocytes, while incubation of NPs with meat extract had no obvious effect on particle adherence. In contrast, contact with intestinal fluid appeared to increase the particle-cell interaction of 20 and 100 nm NPs. Conclusion: Luminal gut-constituents may both attenuate and augment the adherence of NPs to cell surfaces. These effects appear to be dependent on the particle size as well as on the type of interacting protein. While some proteins will rather passivate particles towards cell attachment, possibly by increasing colloid stability or camouflaging attachment sites, certain components of intestinal fluid are capable to modify particle surfaces in such a way that interactions with cellular surface structures result in an increased binding.

Cancers of the gastrointestinal tract are one of the leading causes of death in affluent countries. Although they are usually curable if detected at an early enough stage, the compliance to undergo routine screenings for gastrointestinal cancers in a presymptomatic stage is low, mostly due to the discomfort which current diagnostic measures, such as gastroscopy, colonoscopy, digital rectal exams or feces examinations pose to the patient. Magnetic particle imaging (MPI) has the potential to overcome this hurdle as the procedure is noninvasive, radiation-free and assumed to be more sensitive than most other imaging modalities used today. Yet, MPI bears a catch as it requires special magnetizable contrast agents in the nanoscale range. These contrast agents have to meet a number of requirements. They must be detectable in the magnetic field, be manufacturable in a cost-effective manner, display a certain shelf life, be administrable in a compliant way, be stable in vivo, be specific and sensitive in labeling the neoplastic tissue and must not be harmful to the individual who incorporates them. Due to this complex requirement profile MPI contrast agent engineering lags behind imaging hardware development. In order to close in on this field it is important to identify, rate and define the relevant players which ingested MPI contrast agents have to deal with en route to their destination in the body. Potential players are the anatomy and microanatomy of the gastrointestinal tract, the chemical and biological composition of the luminal constituents as well as potential degradation and scavenger mechanisms. Once the possible impact of these factors has been established contrast agents can be designed to master these challenges. Yet, reaching the destination without causing harm is a necessary but not sufficient requirement. In order to fully exploit the potential of MPI and to qualify it for mass screening the method must be highly sensitive and specific. This may be achieved by targeting systems that direct the contrast agent exclusively and efficiently to the neoplastic tissue in question. In this paper we provide an overview on our current knowledge of the fate of magnetizable contrast agents in the gut, the players which determine this fate, measures to properly endow contrast agents for this task and means to equip them with the necessary specifity and sensitivity.