Fluorescent read out

Usually, the NCE is pipetted together with the en-zyme/substrate complex and the reaction is started with addition of the cofactor solution. Incubation times vary between 15 and 45 min at 37 °C. Afterwards, the reaction is stopped by addition a TRIS/Acetonitile solution and applied to fluorescence read out. 

Additional fluorescence labeled marker substrates with different extension/emission wavelength are on the market e.g. from Invitrogen (www.invitrogen.com) which allows some variation if the NCE/metabolite interfere with the fluorescence read out. 

Many of these dyes and their applications have been reviewed in detail elsewhere (50). Of note, probes are available for both UV and 488 nm excitation and many of these calcium probes (Fura-l, Indo-1, Calcium Green-2) have the advantage of utilizing a ratiometric fluorescence read-out. These dyes have been successfully used in multiparametric analyses, using both flow cytometry and confocal microscopy, which has led to an enhanced understanding of the role of [Ca2+]i in apoptosis. Burchiel et al. (51) have recently published a review of multiparametric flow cytometric Ca2+ analysis. 

FIGURE 44.21 Illustrations of the concept of dynamic arraying showing insertion of the solid phase of different specificity (a) through inlets (A, B... X), trapping of bead clusters using the ultrasonic transducer array (b) and perfusion of sample (c) through inlets (A, B... Y) followed by fluorescence read-out. (Reproduced from Lilliehorn T. et al., Sensors and Actuators B, 106, 851, 2005. With permission.)... 

Schematic representation of the concept of polymeric sensors based on a polymer phase transition as thermoresponsive structure and solvatochromic dyes to provide a visual or fluorescence read-out signal of the change in the polarity of the microenvironment (Pietsch et a ., 2011). (Source Reprinted with permission from the RSC.)... 

Optical read out will compete with micro electrode arrays. New developments in array detectors will open new perspectives. Direct optical detection techniques will add new possibilities to bioanalytical applications in addition to fluorescence measurements presently preferred in biochips read out. 

In conventional chip experiments, fluorescence scanners are used for chip read-out. In the case of laser scanners, HeNe lasers are used as excitation sources and photomultiplier tubes as detectors, whereas CCD-based scanners use white light sources. The optical system can be confocal or non-confocal. Standard biochip experiments are performed using two fluorescent labels as... 

Various methods are used for read-out. Micelle formation and dissociation may be detected by means of a fluorescence probe detect-... 

Fig. 6 (a) Schematic illustration of a flow cytometer used in a suspension array. The sample microspheres are hydrodynamically focused in a fluidic system and read-out by two laser beams. Laser 1 excites the encoding dyes and the fluorescence is detected at two wavelengths. Laser 2 is used to quantify the analyte, (b) Scheme of randomly ordered bead array concept. Beads are pooled and adsorbed into the etched wells of an optical fiber, (c) Scheme of randomly-ordered sedimentation array. A set of encoded microspheres is added to the analyte solution. Subsequent to binding of the analyte, microparticles sediment and assemble at the transparent bottom of a sample tube generating a randomly ordered array. This array is evaluated by microscope optics and a CCD-camera. Reproduced with permission from Refs. [85] and [101]. Copyright 1999, 2008 American Chemical Society... 

CCD cameras have also been used for multichannel fluorescent detection. However, the use of the CCD detector suffers from the need to read out the entire image content information in order to determine the pixel intensity at the flow channel location. Accordingly, a CMOS imager was used. This method offers direct control over individual pixels, and can provide much faster response times and longer integration times for those desired pixels [252]. 

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