ABSTRACT
Free-flow electrophoresis is an ideal tool for preparative separations in continuous microflow. With the approach presented herein for coupling free-flow electrophoresis and mass spectrometry it is now also possible to trace non-fluorescent compounds and identify them by means of mass spectrometry. The functionality of the method and its potential as an integrated separation unit for microflow synthesis is demonstrated by application to a multicomponent [3+2]-cycloannulation.
Subject(s)
Electrophoresis/methods , Mass Spectrometry , Nanotechnology , Benzaldehydes/chemical synthesis , Benzaldehydes/chemistry , Cyclization , Molecular Structure , Organosilicon Compounds/chemical synthesis , Organosilicon Compounds/chemistry , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/chemistryABSTRACT
Herein, we demonstrate the feasibility of a protein-protein interaction analysis and reaction progress monitoring in microfluidic droplets using FRET and microscopic fluorescence lifetime measurements. The fabrication of microdroplet chips using soft- and photolithographic techniques is demonstrated and the resulting chips reliably generate microdroplets of 630 pL and 6.71 nL at frequencies of 7.9 and 0.75 Hz, respectively. They were used for detection of protein-protein interactions in microdroplets using a model system of Alexa Fluor 488 labelled biotinylated BSA, Alexa Fluor 594 labelled streptavidin and unlabelled chicken egg white avidin. These microchips could be used for quantitative detection of avidin and streptavidin in microdroplets in direct and competitive assay formats with nanomolar detection limits, corresponding to attomole protein amounts. Four droplets were found to be sufficient for analytical determination. Fluorescence intensity ratio and fluorescence lifetime measurements were performed and compared for microdroplet FRET determination. A competitive on-chip binding assay for determination of unlabelled avidin using fluorescence lifetime detection could be performed within 135 s only.
Subject(s)
Fluorescence Resonance Energy Transfer , Fluorometry , Microfluidic Analytical Techniques/methods , Protein Interaction Mapping/methods , Avidin/chemistry , Biotin/chemistry , Equipment Design , Microfluidic Analytical Techniques/instrumentation , Microscopy, Confocal , Microscopy, Fluorescence , Protein Binding , Protein Interaction Mapping/instrumentation , Serum Albumin, Bovine/chemistry , Streptavidin/chemistry , Time FactorsABSTRACT
We immobilized adherent human embryonic kidney (HEK) cells--which are able to trace adenosine triphosphate (ATP)--inside a microfluidic free-flow electrophoresis (µFFE) chip in order to develop an integrated device combining separation and biosensing capabilities. HEK 293 cells loaded with fluorescent calcium indicators were used as a model system to enable the spatially and temporally resolved detection of ATP. The local position of a 20 µM ATP stream was successfully visualized by these cells during free-flow electrophoresis, demonstrating the on-line detection capability of this technique towards native, unlabeled compounds.
Subject(s)
Adenosine Triphosphate/analysis , Electrophoresis/instrumentation , Microfluidic Analytical Techniques/instrumentation , Biosensing Techniques/instrumentation , Cell Adhesion , Electrophoresis/methods , Equipment Design , Fluorescent Dyes , HEK293 Cells , Humans , Microfluidic Analytical Techniques/methodsABSTRACT
BACKGROUND: There is an ongoing debate on whether consumers of child pornography pose a risk for hands-on sex offenses. Up until now, there have been very few studies which have analyzed the association between the consumption of child pornography and the subsequent perpetration of hands-on sex offenses. The aim of this study was to examine the recidivism rates for hands-on and hands-off sex offenses in a sample of child pornography users using a 6 year follow-up design. METHODS: The current study population consisted of 231 men, who were subsequently charged with consumption of illegal pornographic material after being detected by a special operation against Internet child pornography, conducted by the Swiss police in 2002. Criminal history, as well as recidivism, was assessed using the criminal records from 2008. RESULTS: 4.8% (n = 11) of the study sample had a prior conviction for a sexual and/or violent offense, 1% (n = 2) for a hands-on sex offense, involving child sexual abuse, 3.3% (n = 8) for a hands-off sex offense and one for a nonsexual violent offense. When applying a broad definition of recidivism, which included ongoing investigations, charges and convictions, 3% (n = 7) of the study sample recidivated with a violent and/or sex offense, 3.9% (n = 9) with a hands-off sex offense and 0.8% (n = 2) with a hands-on sex offense. CONCLUSION: Consuming child pornography alone is not a risk factor for committing hands-on sex offenses - at least not for those subjects who had never committed a hands-on sex offense. The majority of the investigated consumers had no previous convictions for hands-on sex offenses. For those offenders, the prognosis for hands-on sex offenses, as well as for recidivism with child pornography, is favorable.