Microfluidic enzyme-sensors

Immunoaffinity chromatography (IAC), 6 400—402 12 137, 145 Immunoanalyzers, automated, 14 150 Immunoassay(s), 14 135-159. See also Immunoassay- DNA probe hybrid assays Immunoassay methods Immuno(bio)sensors antibody-antigen reaction, 14 136-138 basic technology in, 14 138-140 chemiluminescent, 14 150-151 classification of, 14 140-153 design of, 14 139-140 enzyme, 14 143-148 fluorescence, 14 148-150 highly specific, 14 153 historical perspective on, 14 136 microarrays and, 14 156—157 microfluidics in, 26 968—969 monoclonal versus polyclonal antibodies in, 14 152-153... 

FIGURE 10.17 Schematic of microfluidic device consisting of an H-filter for cell lysis/fractionation coupled to a T-sensor for detection of an intracellular enzyme (( -galactosidase). Pump rates are controlled at all inlets and one outlet. In the H-filter (left), lytic agent diffuses into the cell suspension, and lyses the cells. Intracellular components (enzyme) then diffuse away from the cell stream and some are brought around the corner into the detection channel. In the T-sensor (right), the presence of (1-Gal is detected fluorescently [1051]. Reprinted with permission from the American Chemical Society. 

Kurita, R., Hayashi, K., Fan, X., Yamamoto, K., Kato, T., Niwa, O., Microfluidic device integrated with pre-reactor and dual enzyme-modified microelectrodes for monitoring in vivo glucose and lactate. Sensors Actuators B 2002, 87, 296-303. 

An example of the immobilization of antibodies on channel surfaces was presented by Eteshola and Leckband [395]. A microfluidic sensor chip was developed to quantify a model analyte (sheep IgM) with sensitivities down to 17 nM. This was achieved by first immobilizing a layer of bovine serum albumine (BSA) onto the channel wall, followed by specific adsorption of protein A to which the primary antibody for IgM was coupled covalently. This antibody could capture IgM, which was detected with the secondary antibody, labeled with horseradish peroxidase (Scheme 4.91). This enzyme catalyzes the conversion of the fluorogenic substrate 3-(p-hydroxyphenyl)propioni c acid into a fluorophore, which was quantified off-chip with a spectrofluorometer. The measured fluorescence signal was proportional to the analyte concentration in the test sample. 

The first type of enzyme application in microfluidics is chemical sensing. Sensors can be constmeted in cases where an enzyme turns over a particular smaU-molecule substrate to produce a product quantifiable by fluorescence, chemiluminescence, absorbance spectroscopy, or electrochemical detectors. In cases where the substrate is not detectable itself, an enzymatic product can often be coupled to another enzyme that produces a detectable product. For example, there are a wide variety of small molecules (such as nutrients, amino acids, and sugars) that can be coupled to the chemiluminescent reaction of luminol and peroxide in the presence of horseradish peroxidase. These enzyme-substrate assays were the first to be adapted to microfluidic devices — a great number of small-molecule sensors have been developed based on microfluidic channels with electroosmotic or hydrodynamic flow, and pre-loaded microfluidic cartridges containing nanoliter volumes of reagents have... 

Fig. 1.13 Calibration of 8-sensor microfluidic array with off-line analyte capture by multilabel HRP-MP-Ab2 particles using 200,000 HRP labels/particle for PSA and IL-6 mixtures in serum. Signals developed by injecting 1 mM hydroquinone mediator + 100 pM hydrogen peroxide as enzyme activator, c Simultaneous determinations by the array compared to individual ELIS As for PSA and IL-6 in patient serum 1-4 are from prostate cancer patients 5 is a cancer-free control. Reproduced with permission from ref. 112 copyright Elsevier, 2011...

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