M any components in biological matrices influence the result of an analysis, affecting assay sensitivity and reproducibility. Improved matrix management becomes critical as requirements for higher assay sensitivity and increased process throughput become more demanding. There are several robotic laboratory automation systems that are commercially available, which serve to minimize matrix interference by performing purification and extraction protocols. However, there is an unmet need of inline matrix effect reduction solutions to reduce the processing time and cost for automated sample preparation. In microfluidics, effective matrix management is essential for developing fully integrated systems capable of meeting these requirements. This review surveys current biological matrix management techniques for liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and binding assays with a view toward building automatable processes. For some systems, simple sample-preparation methods, such as dilution and protein precipitation (PPT), are sufficient, whereas other systems require labor-intensive methods, such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE). To achieve high throughput, PPT, LLE, and SPE have been adopted to 96-well-plate format. Online SPE has also been coupled with LC-MS/MS to automate sample preparation and analysis of urine, plasma, and serum matrices. However, offline processing of whole blood is still required to obtain plasma and serum. The ultimate goal of implementing sample preparation to reduce matrix effects within untreated sample is to achieve reproducibility and sensitivity required by the application; therefore, inline sample preparation integrated with molecular analysis will be highly significant for laboratory automation. Electrokinetic methods have the potential of handling whole-blood, urine, and saliva samples and can be incorporated into microfluidic systems for full automation. Optimization of analysis conditions and the use of appropriate standards have likewise assisted in reducing or correcting matrix effects and will also be discussed. ( JALA 2010;15:233-42)

A convenient homogeneous enzyme immunoassay for estradiol is described. Unlike heterogeneous immunoassays, which require time-consuming separation steps or expensive automated systems, homogeneous immunoassays, wherein all reagents are freely suspended in bulk solution, can be simple and fast without costly instrumentation. The key component of this assay system, an estradiol-reporter enzyme conjugate, was prepared by covalently binding β-estradiol-6-(O-carboxymethyl)oxime to glucose-6-phosphate dehydrogenase (G6PDH) by an N-hydroxysuccinimide-enhanced, carbodiimide-mediated coupling reaction. The estradiol-G6PDH activity can be repressed up to 46% upon anti-estradiol antibody binding. The lower detection limit of the assay is 1 nM estradiol in aqueous solution, and the standard curve is linear on logit-log scale-up to 6.7 µM estradiol. A detection limit of 11.5 nM in estradiol-spiked human serum samples suggests the feasibility of applying this assay to monitor estradiol levels for the prediction and prevention of ovarian hyperstimulation syndrome.

A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. Toward understanding the antibody-induced inhibition mechanism, characterization of morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates was pursued. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine-3-glucuronide NHS-ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially, the cause of activity loss due to the coupling reaction.

A practical approach for constructing enzyme-multiplied immunoassay technique (EMIT)-based protein/peptide assays is described. Normally used in small-molecule drug testing, EMIT is a homogeneous assay method that is attractive for its simplicity, sensitivity, and rapidity. The EMIT-based peptide/protein assay was developed by conjugating a cysteine-modified HA peptide (from influenza hemagglutinin A) to the reporter enzyme, glucose-6-phosphate dehydrogenase. The 13-min assay gave a free HA limit of detection of 10 nM and proved effective for detection of a high-molecular-weight model protein tagged with HA. Similar EMIT-based assay approaches may be developed for applications in biotoxin and infectious disease detection.