Optical detection protocol

Figure 19.16 Optical detection protocol using displacement assays in bio-inspired mesoporous systems.

FIGURE 4. Optical detection protocol using displacement assays in bioinspired mesoporous systems, (a) Functionalization of mesoporous walls with selected binding sites, (b) Loading of functionalized material with certain dye. (c) Displacement of the dye with target anion. 

Of the analytical procedures used for the determination of LAS in soils (Table 6.7.1), most methods rely on (Soxhlet) extraction with methanol, followed by clean-up on SPE cartridges (RP-C18 and/or SAX) and final quantitative measurements by HPLC—UV/FL. Applying this protocol, detection limits were achieved ranging between 0.05 and 5 mg kg-1 depending on the matrix, the enrichment factor and the optical detection system employed. 

This chapter provides an overview of the analysis of TPs in the environment of three important classes of synthetic chemicals namely pesticides, human and veterinary pharmaceuticals, and personal care products (PPCPs). A series of analytical protocols applied to determine and analyze TPs of manmade chemicals originating from photolysis as well as from microbial degradation in the environment and wastewater treatment plants (WWTPs) is presented. Furthermore, strategies for identifying unknown TPs of xenobi-otic compounds including pesticides and PPCPs are presented based on the combination of mass spectrometric techniques and NMR, IR, and optical detection systems like DAD and FL. 

Alternative 2. By hyphenating an optical fiber sensor for implementing disk-based solid-phase extraction and direct optosensing at the solid surface, there is no need for further elution to perform the optical detection in the eluate phase, as demanded in conventional sorbent-extraction protocols previously reported for sulfide, thus yielding improved enrichment factors [14] (Figure 7.5). 

In the first LDMS-based detection of malaria in human subjects (unpublished), lOOpl P. falciparum or P. v/vax-infected blood samples, grouped into three different parasitemia ranges—low (10-150 parasites/pl), mid (2 x 103 parasites/pl), and high (25 x 103-60 x 103 parasites/pl)—have been examined using both sample preparation protocols. Parasitemia levels in these samples were previously determined independently for each sample by optical microscopy examination of blood smears. The LDMS data clearly indicate that... 

BRET [31, 32]), lock-in detection techniques exploiting optical switches [33], and schemes for alternating D/A excitation (ALEX [34]). The increased attention to quantitative FRET imaging encompasses the use of polarization [35-39], the perennial issue of calibration and standards [40-44], and practical guides to operational principles and protocols ([45, 46] and other references above). The fundamental distinctions between the requirements for live and fixed cell imaging cannot be overemphasized, as is exemplified in a report of erroneous FRET determinations with visible fluorescent proteins (VFPs) in fixed cells [47],... 

Sensitivity impacts upon the limit of detection and resolution of the device, making it a key performance parameter. Recently, several strategies have been developed in order to provide sensitivity enhancements for optical sensor platforms based on both optical absorption and fluorescence phenomena. These strategies are the result of rigorous theoretical analyses of the relevant systems and, combined with polymer processing technology and planar fabrication protocols, provide a viable route for the development of low-cost, efficient optical sensor platforms. 

However, luminescence-based detection techniques often require a high number of steps. Consider ELISA as an example. As a first step, the sample is introduced into a 96-well plate an antibody targeting the antigen of interest has been immobilized to the wells of the plate. After a rinse, the wells contain the antibody and any bound antigen. However, although the antigen has been isolated, the protocol is nowhere near completion. The remaining steps include another antibody (different from the first) to form a sandwich assay, a secondary antibody with an enzymatic label, and a substrate that is luminescent when activated by the enzyme. Finally, the sample is analyzed by relatively expensive detection optics to determine the amount of analyte that was captured in the assay. The steps are illustrated in Fig. 14.1a. 

In a modern dew-point instrument, a sample is equilibrated within the headspace of a sealed chamber containing a mirror, an optical sensor, an internal fan, and an infrared thermometer (Figure A2.2.2). At equilibrium, the relative humidity of the air in the chamber is the same as the water activity of the sample. A thermoelectric (Peltier) cooler precisely controls the mirror temperature. An optical reflectance sensor detects the exact point at which condensation first appears a beam of infrared light is directed onto the mirror and reflected back to a photodetector, which detects the change in reflectance when condensation occurs on the mirror. A thermocouple attached to the mirror accurately measures the dew-point temperature. The internal fan is for air circulation to reduce vapor equilibrium time and to control the boundary layer conductance of the mirror surface (Campbell and Lewis, 1998). Additionally, an infrared thermometer measures the sample surface temperature. Both the dew-point and sample temperatures are then used to determine the water activity. The range of a commercially available dew-point meter is 0.030 to 1.000 aw, with a resolution of 0.001 aw and accuracy of 0.003 aw. Measurement time is typically less than 5 min. The performance of the instrument should be routinely verified as described in the Support Protocol. 

The present chapter critically encompasses developments and achievements reached in MIP-based selective sensing combined with optical, piezoelectric (PZ) and electrochemical signal transduction. General procedures of MIP preparation along with methods of MIP immobilization for chemosensor fabrication are presented. Protocols of analyte determination involving measurement complexities, like template presence or absence, have been addressed in detail. Moreover, analytical parameters, such as detectability, sensitivity, selectivity, linear dynamic... 

Important features to consider are the ease of software use and the ability to change the optical filters depending upon the peak absorption and emission spectra of the labels used. The dynamic range and sensitivity of the readers are important factors that can affect the assay protocol. A protocol that is suitable for one type of reader may not work as well in a second type of reader due to variations in the optics resulting in differences in dynamic range and sensitivity. Sensitivity is important because it aids in the miniaturization of assays and helps reduce the amounts of expensive reagents required. The ability to detect small quantities of product in an enzyme reaction means that less enzyme will be required. 

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