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Background There is increasing evidence that Parkinson’s disease (PD) might start in the gut, thus involving and compromising also the enteric nervous system (ENS). At the clinical onset of the disease the majority of dopaminergic neurons in the midbrain is already destroyed, so that the lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use a transgenic A30P-α-synuclein-overexpressing PD mouse model to identify appropriate candidate markers in the gut before hallmark symptoms begin to manifest. Methods Based on a gait analysis and striatal dopamine levels, we defined 2-month-old A30P mice as pre-symptomatic (psA30P), since they are not showing any motoric impairments of the skeletal neuromuscular system and no reduced dopamine levels, but an intestinal α-synuclein pathology. Mice at this particular age were further used to analyze functional and molecular alterations in both, the gastrointestinal tract and the ENS, to identify early pathological changes. We examined the gastrointestinal motility, the molecular composition of the ENS, as well as the expression of regulating miRNAs. Moreover, we applied A30P-α-synuclein challenges in vitro to simulate PD in the ENS. Results A retarded gut motility and early molecular dysregulations were found in the myenteric plexus of psA30P mice. We found that i.e. neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are significantly altered in the psA30P, thus representing potential biomarkers for early PD. Many of the dysregulated miRNAs found in the psA30P mice are reported to be changed in PD patients as well, either in blood, cerebrospinal fluid or brain tissue. Interestingly, the in vitro approaches delivered similar changes in the ENS cultures as seen in the transgenic animals, thus confirming the data from the mouse model. Conclusions These findings provide an interesting and novel approach for the identification of appropriate biomarkers in men.

Chromatin condensation is one of the main factors essential for sperm function. Evaluation of chromatin condensation by current methods render the assessed sperm unsuitable for assisted reproduction. We examined the Raman spectra of normal morphology sperm to determine whether a non-invasive confocal Raman spectroscopy can detect spectral differences between groups having different levels of chromatin condensation. Semen samples from 85 donors who underwent ICSI were obtained. Chromomycin A3, aniline blue and acridine orange staining were performed to evaluate the protamine deficiency, histone retention and DNA fragmentation respectively. Raman spectra were obtained from 50 normal morphology sperm for each donor. Spectral analysis was performed using home written programs in LabVIEW software and samples were grouped based on chromomycin A3 staining. Raman peaks intensities at 670 cm-1, 731 cm-1, 785 cm-1, 858 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 were significantly correlated with at least one of the sperm staining methods. The median intensity of the Raman peaks at 670 cm-1, 731 cm-1, 785 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 show a significant difference between the CMA3≤41 and CMA3>41groups. The Raman spectroscopic measurements represent a promising diagnostic tool that has the ability to label-free detect sperm with chromatin abnormalities, such as improper chromatin condensation and DNA fragmentation to a certain degree similar to that of the existing staining techniques at the individual cell level.

Peptidic radiotracers are highly potent substances for the specific in vivo imaging of various biological targets with Single Photon Emission Computed Tomography and Positron Emission Tomography. However, some radiolabeled peptides such as bombesin analogs were shown to exhibit only a limited stability, hampering a successful target visualization. One option to positively influence the stability of radiolabeled peptides is the introduction of certain artificial molecular scaffolds. In order to comparatively assess the influence of different structure elements on the stability of radiolabeled peptides and to identify those structure elements being most useful for peptide radiotracer stabilization, several monomeric and dimeric bombesin derivatives were synthesized, exhibiting differing molecular designs and the chelator NODAGA for (68) Ga-labeling. The radiolabeled peptides were evaluated regarding their in vitro stability in human serum to determine the influence of the introduced molecular scaffolds on the peptides' serum stabilities. The results of the evaluations showed that the introduction of scaffold structures and the overall molecular design have a substantial impact on the stabilities of the resulting peptidic radiotracers. But besides some general trends found using certain scaffold structures, the obtained results point to the necessity to empirically assess their influence on stability for each susceptible peptidic radiotracer individually.

Autoradiolysis describes the degradation of radioactively labeled compounds due to the activity of the labeled compounds themselves. It scales with activity concentration and is of importance for high activity and microfluidic PET tracer synthesis. This study shows that microfluidic devices can be shaped to reduce autoradiolysis by geometric exclusion of positron interaction. A model is developed and confirmed by demonstrating in-capillary storage of non-stabilized [18F]FDG (2-[18F]Fluoro-2-deoxy- d -glucose) at max. 23 GBq/ml while maintaining > 90 % radiochemical purity over 14 h.

The presented paper describes a magnetic field sensor system for the real-time monitoring of magnetic resonance (MR) image encoding in the presence of gradient and main magnetic field imperfections. For this purpose, a novel simplified design for nuclear magnetic resonance (NMR) probes has been developed. The design fulfills the requirements of providing below ppm measurement accuracy in combination with long readout times. To this end, NMR is performed with a nanoliter water volume, which together with the radiofrequency (RF) solenoid coil is cast into an Epoxy holder. Based on the Larmor relation (i.e. the proportionality between the NMR resonance frequency and the local B 0 ), time varying magnetic field information is extracted from the phase evolution of the detected RF signals. Susceptibility matching techniques are applied to homogenize the B 0 field across the NMR sample and in this way to extend the readout time of the NMR probes. The presented design of NMR probe is significantly simpler compared to previous presented ones by De Zanche et al. in the sense that it omits the glass capillary plus additional matching fluidics. The optimization of the susceptibility for the Epoxy is described and results of the probe characterization measurements are presented. The feasibility of magnetic field monitored MRI is demonstrated for ultra-fast single- and multishot data acquisition schemes.

Peptidic radiotracers are highly potent substances for the specific in vivo imaging of various biological targets with Single Photon Emission Computed Tomography and Positron Emission Tomography. However, some radiolabeled peptides such as bombesin analogs were shown to exhibit only a limited stability, hampering a successful target visualization. One option to positively influence the stability of radiolabeled peptides is the introduction of certain artificial molecular scaffolds. In order to comparatively assess the influence of different structure elements on the stability of radiolabeled peptides and to identify those structure elements being most useful for peptide radiotracer stabilization, several monomeric and dimeric bombesin derivatives were synthesized, exhibiting differing molecular designs and the chelator NODAGA for (68) Ga-labeling. The radiolabeled peptides were evaluated regarding their in vitro stability in human serum to determine the influence of the introduced molecular scaffolds on the peptides' serum stabilities. The results of the evaluations showed that the introduction of scaffold structures and the overall molecular design have a substantial impact on the stabilities of the resulting peptidic radiotracers. But besides some general trends found using certain scaffold structures, the obtained results point to the necessity to empirically assess their influence on stability for each susceptible peptidic radiotracer individually.

The application of microfluidics to the synthesis of Positron Emission Tomography (PET) tracers has been explored for more than a decade. Microfluidic benefits such as superior temperature control have been successfully applied to PET tracer synthesis. However, the design of a compact microfluidic platform capable of executing a complete PET tracer synthesis workflow while maintaining prospects for commercialization remains a significant challenge. This study uses an integral system design approach to tackle commercialization challenges such as the material to process compatibility with a path towards cost effective lab-on-chip mass manufacturing from the start. It integrates all functional elements required for a simple PET tracer synthesis into one compact radiochemistry platform. For the lab-on-chip this includes the integration of on-chip valves, on-chip solid phase extraction (SPE), on-chip reactors and a reversible fluid interface while maintaining compatibility with all process chemicals, temperatures and chip mass manufacturing techniques. For the radiochemistry device it includes an automated chip-machine interface enabling one-move connection of all valve actuators and fluid connectors. A vial-based reagent supply as well as methods to transfer reagents efficiently from the vials to the chip has been integrated. After validation of all those functional elements, the microfluidic platform was exemplarily employed for the automated synthesis of a Gastrin-releasing peptide receptor (GRP-R) binding the PEGylated Bombesin BN(7-14)-derivative ([(18)F]PESIN) based PET tracer.

We present a chemical sensor based on a microfabricated array of eight silicon cantilevers actuated at their resonance-frequency and functionalized by polymer coatings. The operating principle relies on transduction of chemical or physical processes into a mechanical response. After exposure to analyte vapor, analyte molecules diffuse into the cantilever coating, which begins to swell. Jointly with the mass increase, a change of interfacial stress between coating and cantilever occurs, resulting in a bending of the cantilevers. Our setup allows the simultaneous detection of cantilever oscillation and bending of eight cantilevers by time-multiplexed optical beam deflection readout. The ac component of the cantilever response is demodulated, and the cantilever resonance-frequency is tracked by a custom-built phase-locked loop. By filtering out the ac component (oscillation), the dc signal (bending) is extracted, yielding information on mass as well as surface stress changes simultaneously. Detection results of water, primary alcohols, alkanes and perfumes are presented.

A novel chemical sensor based on a micromechanical array of silicon cantilevers is presented. Chemical reactions are transduced by sensitization of cantilevers with coatings such as metals, self-assembled monolayers, or polymers into a mechanical response. This is read out using an optical beam-deflection technique by a sequential readout scheme. Reference cantilever sensors permit subtraction of background signals (differential measurement). Coating of each cantilever sensor with a different sensitive layer allows operation of the array-device as a new form of chemical nose. Detection of hydrogen, primary alcohols, natural flavors, and water vapor is demonstrated. We show that the magnitude of sensor response is proportional to the amount of analyte present.

An array of microcantilever beams, coated with a self-assembled monolayer of bistable, redox-controllable [3]rotaxane molecules, undergoes controllable and reversible bending when it is exposed to chemical oxidants and reductants. Conversely, beams that are coated with a redox-active but mechanically inert control compound do not display the same bending. A series of control experiments and rational assessments preclude the influence of heat, photothermal effects, and pH variation as potential mechanisms of beam bending. Along with a simple calculation from a force balance diagram, these observations support the hypothesis that the cumulative nanoscale movements within surface-bound “molecular muscles” can be harnessed to perform larger-scale mechanical work.

The interfacial stress of self-assembled monolayers on Au exposed to buffers of various pH values and ionic strengths is measured as a function of the liquid environment. The method uses two thiol-modified Au-covered silicon cantilevers and a differential method to compensate for thermal and refractive index changes of the liquid environment. Increasing pH and ionic strength leads to a bending, i.e., a compressive stress, of a mercaptohexadecanoic acid-covered cantilever compared to a hexadecanethiol-covered reference cantilever. In addition, the interfacial stress is found to be highly dependent on the surface density of the ionizeable mercaptohexadecanoic acid molecules when they are coadsorbed with hexadecanethiols on Au.

Raman detection of nitrogen gas is very difficult without a multi-pass arrangement and high laser power. Hollow-core photonic bandgap fibers (HC-PBF) provide an excellent means of concentrating light energy in a very small volume and long interaction path between gas and laser. One particular commercial fiber with a core diameter of 4.9 microns offers losses of about 1dB/m for wavelengths between 510 and 610 nm. If 514nm laser is used for excitation, the entire Raman spectrum up to above 3000 cm -1 will be contained within the transmission band of the fiber. A standard Raman microscope launches mW level 514nm laser light into the PBF and collects backscattered Raman signal exiting the fiber. The resulting spectra of nitrogen gas in air at ambient temperature and pressure exhibit a signal enhancement of about several thousand over what is attainable with the objective in air and no fiber. The design and fabrication of a flow-through cell to hold and align the fiber end allowed the instrument calibration for varying concentrations of nitrogen. The enhancement was also found to be a function of fiber length. Due to the high achieved Raman signal, rotational spectral of nitrogen and oxygen were observed in the PBF for the first time to the best of our knowledge.

Two switchable, palindromically constituted bistable [3]rotaxanes have been designed and synthesized with a pair of mechanically mobile rings encircling a single dumbbell. These designs are reminiscent of a "molecular muscle" for the purposes of amplifying and harnessing molecular mechanical motions. The location of the two cyclobis(paraquat-p-phenylene) (CBPQT(4+)) rings can be controlled to be on either tetrathiafulvalene (TTF) or naphthalene (NP) stations, either chemically ((1)H NMR spectroscopy) or electrochemically (cyclic voltammetry), such that switching of inter-ring distances from 4.2 to 1.4 nm mimics the contraction and extension of skeletal muscle, albeit on a shorter length scale. Fast scan-rate cyclic voltammetry at low temperatures reveals stepwise oxidations and movements of one-half of the [3]rotaxane and then of the other, a process that appears to be concerted at room temperature. The active form of the bistable [3]rotaxane bears disulfide tethers attached covalently to both of the CBPQT(4+) ring components for the purpose of its self-assembly onto a gold surface. An array of flexible microcantilever beams, each coated on one side with a monolayer of 6 billion of the active bistable [3]rotaxane molecules, undergoes controllable and reversible bending up and down when it is exposed to the synchronous addition of aqueous chemical oxidants and reductants. The beam bending is correlated with flexing of the surface-bound molecular muscles, whereas a monolayer of the dumbbell alone is inactive under the same conditions. This observation supports the hypothesis that the cumulative nanoscale movements within surface-bound "molecular muscles" can be harnessed to perform larger-scale mechanical work.

We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ . Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3′ and 5′ overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.

We report the specific transduction, via surface stress changes, of DNA hybridization and receptor-ligand binding into a direct nanomechanical response of microfabricated cantilevers. Cantilevers in an array were functionalized with a selection of biomolecules. The differential deflection of the cantilevers was found to provide a true molecular recognition signal despite large nonspecific responses of individual cantilevers. Hybridization of complementary oligonucleotides shows that a single base mismatch between two 12-mer oligonucleotides is clearly detectable. Similar experiments on protein A–immunoglobulin interactions demonstrate the wide-ranging applicability of nanomechanical transduction to detect biomolecular recognition.

We present quantitative and qualitative detection of analyte vapors using a microfabricated silicon cantilever array. To observe transduction of physical and chemical processes into nanomechanical motion of the cantilever, swelling of a polymer layer on the cantilever is monitored during exposure to the analyte. This motion is tracked by a beam-deflection technique using a time multiplexing scheme. The response pattern of eight cantilevers is analyzed via principal component analysis (PCA) and artificial neural network (ANN) techniques, which facilitates the application of the device as an artificial chemical nose. Analytes tested comprise chemical solvents, a homologous series of primary alcohols, and natural flavors. First differential measurements of surface stress change due to protein adsorption on a cantilever array are shown using a liquid cell.

We present a novel chemical sensor based on a microfabricated array of silicon cantilevers. Individual cantilevers are sensitized for the detection of analytes using metal coatings. Analyte molecules chemisorbing or physisorbing on the cantilever coating and chemical reactions produce a change in interfacial stress between analyte molecules and cantilever. This leads to a nanomechanical response of the cantilever, i.e. bending. The bending is read out using a time-multiplexed optical beam-deflection technique. From magnitude and temporal evolution of the bending, quantitative information on analyte species and concentration is derived. Here, we demonstrate the detection of ethene and water vapor with such a nanomechanical nose.

We present a novel chemical sensor based on a micromechanical array of silicon cantilevers sensitized for the detection of analytes using cantilever coatings such as metals, self-assembled monolayers, or polymers. Chemical reactions are transduced into a mechanical response and read out using an optical beam-deflection technique. Detection of primary alcohols, natural flavors, and water vapor is demonstrated.