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Most biochemical reactions depend on the pH value of the aqueous environment and some are strongly favoured to occur in an acidic environment. A non-invasive control of pH to tightly regulate such reactions with defined start and end points is a highly desirable feature in certain applications, but has proven difficult to achieve so far. We report a novel optical approach to reversibly control a typical biochemical reaction by changing the pH and using acid phosphatase as a model enzyme. The reversible photoacid G-acid functions as a proton donor, changing the pH rapidly and reversibly by using high power UV LEDs as an illumination source in our experimental setup. The reaction can be tightly controlled by simply switching the light on and off and should be applicable to a wide range of other enzymatic reactions, thus enabling miniaturization and parallelization through non-invasive optical means.
Pneumonia is one of the most common and potentially lethal infectious conditions worldwide. Streptococcus pneumoniae is the pathogen most frequently associated with bacterial community-acquired pneumonia, while Legionella pneumophila is the major cause for local outbreaks of legionellosis. Both pathogens can be difficult to diagnose since signs and symptoms are nonspecific and do not differ from other causes of pneumonia. Therefore, a rapid diagnosis within a clinically relevant time is essential for a fast onset of the proper treatment. Although methods based on polymerase chain reaction significantly improved the identification of pathogens, they are difficult to conduct and need specialized equipment. We describe a rapid and sensitive test using isothermal recombinase polymerase amplification and detection on a disposable test strip. This method does not require any special instrumentation and can be performed in less than 20 min. The analytical sensitivity in the multiplex assay amplifying specific regions of S. pneumoniae and L. pneumophila simultaneously was 10 CFUs of genomic DNA per reaction. In cross detection studies with closely related strains and other bacterial agents the specificity of the RPA was confirmed. The presented method is applicable for near patient and field testing with a rather simple routine and the possibility for a read out with the naked eye.
The use of alternating current (AC) electrokinetic forces, like dielectrophoresis and AC electroosmosis, as a simple and fast method to immobilize sub-micrometer objects onto nanoelectrode arrays is presented. Due to its medical relevance, the influenza virus is chosen as a model organism. One of the outstanding features is that the immobilization of viral material to the electrodes can be achieved permanently, allowing subsequent handling independently from the electrical setup. Thus, by using merely electric fields, we demonstrate that the need of prior chemical surface modification could become obsolete. The accumulation of viral material over time is observed by fluorescence microscopy. The influences of side effects like electrothermal fluid flow, causing a fluid motion above the electrodes and causing an intensity gradient within the electrode array, are discussed. Due to the improved resolution by combining fluorescence microscopy with deconvolution, it is shown that the viral material is mainly drawn to the electrode edge and to a lesser extent to the electrode surface. Finally, areas of application for this functionalization technique are presented.
The application of inhomogeneous AC electric fields for molecular immobilization is a very fast and simple method that does not require any adaptions to the molecule's functional groups or charges. Here, the method is applied to a completely new category of molecules: small organic fluorescence dyes, whose dimensions amount to only 1 nm or even less. The presented setup and the electric field parameters used allow immobilization of dye molecules on the whole electrode surface as opposed to pure dielectrophoretic applications, where molecules are attracted only to regions of high electric field gradients, i.e., to the electrode tips and edges. In addition to dielectrophoresis and AC electrokinetic flow, molecular scale interactions and electrophoresis at short time scales are discussed as further mechanisms leading to migration and immobilization of the molecules.
Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500 nm diameter and 20 nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60 fg HRP by observing the enzymatic turnover of Amplex Red and H2O2 to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications.