Microfluidic analysis techniques for safety assessment of pharmaceutical nano- and microsystems

This chapter reviews the evolution of microfabrication methods and materials, applicable to manufacturing of micro total analysis systems (or lab‐on‐a‐chip), from a general perspective. It discusses the possibilities and limitations associated with microfluidic cell culturing, or so called organ‐on‐a‐chip technology, together with selected examples of their exploitation to characterization of pharmaceutical nano‐ and microsystems. Materials selection plays a pivotal role in terms of ensuring the cell adhesion and viability as well as defining the prevailing culture conditions inside the microfluidic channels. The chapter focuses on the hepatic safety assessment of nanoparticles and gives an overview of the development of microfluidic immobilized enzyme reactors that could facilitate examination of the hepatic effects of nanomedicines under physiologically relevant conditions. It also provides an overview of the future prospects regarding system‐level integration possibilities facilitated by microfabrication of miniaturized separation and sample preparation systems as integral parts of microfluidic in vitro models.Non peer reviewe

A Novel Methylation PCR that Offers Standardized Determination of FMR1 Methylation and CGG Repeat Length without Southern Blot Analysis

Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%–100%) with an accuracy of 99% (95% CI: 93%–100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories