This review offers an overview of the relatively young research area of continuous flow lab-on-a-chip for synthetic applications. A short introduction on the basic aspects of lab-on-a-chip is given in the first part. Subsequently, the effects of downscaling reaction vessels as well as the advantages of the continuous flow microfluidic approach over conventional chemical laboratory batch methodologies are illustrated by a number of examples of organic reactions carried out in microfluidic devices. The last part deals with a key issue of the lab-on-a-chip approach, viz. the integration of the microreactor with the analytical instrumentation to achieve high-throughput reaction monitoring.

A continuous flow micro total analysis system (micro-TAS) consisting of an on-chip microfluidic device connected to a matrix assisted laser desorption ionization [MALDI] time-of-flight [TOF] mass spectrometer (MS) as an analytical screening system is presented. Reaction microchannels and inlet/outlet reservoirs were fabricated by powderblasting on glass wafers that were then bonded to silicon substrates. The novel lab-on-a-chip was realized by integrating the microdevice with a MALDI-TOFMS standard sample plate used as carrier to get the microfluidic device in the MALDI instrument. A novel pressure-driven pumping mechanism using the vacuum of the instrument as a driving force induces flow in the reaction microchannel in a self-activating way. Organic syntheses as well as biochemical reactions are carried out entirely inside the MALDI-MS ionization vacuum chamber and analyzed on-line by MALDI-TOFMS in real time. The effectiveness of the micro-TAS system has been successfully demonstrated with several examples of (bio)chemical reactions.

The integration of a monitoring port along the microfluidic path of a MALDI-chip integrated device is described. Optimization of the microreactor design allows longer reaction and measuring times. The Schiff base reaction between 4-tert-butylaniline (1) and 4-tert-butylbenzaldehyde (2) in ethanol was carried out on-chip in the MALDI ionization chamber and the formed imine 3 was detected in real time, demonstrating the feasibility of the "monitoring window" approach. This preliminary result opens the way to on-chip kinetic studies by MALDI-MS, by opening multiple monitoring windows along the microchannel.

This report presents and describes a simple and scalable method for producing functional DNA microarrays within enclosed polymeric, PMMA, microfluidic devices. Brief (30 s) exposure to UV simultaneously immobilized poly(T)poly(C)-tagged DNA probes to the surface of unmodified PMMA and activated the surface for bonding below the glass transition temperature of the bulk PMMA. Functionality and validation of the enclosed PMMA microarrays was demonstrated as 18 patients were correctly genotyped for all eight mutation sites in the HBB gene interrogated. The fabrication process therefore produced probes with desired hybridization properties and sufficient bonding between PMMA layers to allow construction of microfluidic devices. The streamlined fabrication method is suited to the production of low-cost microfluidic microarray-based diagnostic devices and, as such, is equally applicable to the development of diagnostics for both resource rich and resource limited settings.

Surface phenomena are an important contribution to the "chip effect", leading to higher yields and shorter reaction times, as demonstrated for the acid-catalysed esterification of 9-pyrenebutyric acid within a glass fabricated micro reactor.

The reaction of propyl isocyanate (2), benzyl isocyanate (3), and toluene-2,4-diisocyanate (4) with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (1) to yield the corresponding urea derivatives 5 was carried out in a continuous flow glass microfluidics chip. Real-time monitoring of the derivatization reactions was done by electrospray ionization mass spectrometry, making use of a recently reported modular chip-MS interface. Rate constants of 1.5 x 10(4), 5.2 x 10(4), and 2.4 x 10(4) M(-1) min(-1) were determined for 2, 3, and 4, respectively. Using macroscale batch conditions, the rate constants are 3-4 times lower. The faster on-chip kinetics is attributed to the more efficient molecular diffusion in the micrometer-sized channel.

Two simple interfaces were designed and realized, enabling on-line coupling of microfluidics reactor chips to a nanoflow electrospray ionization (NESI) time-of-flight (TOF) mass spectrometer (MS). The interfaces are based on two different approaches: a monolithically integrated design, in which ionization is assisted by on-chip gas nebulization, and a modular approach implying the use of commercially available Picospray tips. Using reserpine as a reference compound in a 1ratio1 mixture of acetonitrile and water revealed that both interfaces provide a remarkably stable mass spectrometric signal (standard deviations lower than 8% and 1% for the monolithic and modular approaches, respectively). Glass microreactors, containing mixing zones, were fabricated and coupled to the modular interface with perfluoroelastomer Nanoport fluidics connectors, providing a tool to study chemical reactions on-line. Investigation of the mixing dynamics showed that complete on-chip reagents mixing is achieved within a few tens of milliseconds. Metal-ligand interactions of Zn-porphyrin with pyridine (2), 4-ethylpyridine (3), 4-phenylpyridine (4), N-methylimidazole (5), and N-butylimidazole (6) in acetonitrile as well as host-guest complexations of beta-cyclodextrin (7) with N-(1-adamantyl)acetamide (8) or 4-tert-butylacetanilide (9) in water were studied by mass spectrometry using the modular NESI-chip interface. From on-chip dilution-based mass spectrometric titrations of Zn-porphyrin 1 with pyridine (2) or 4-phenylpyridine (4) in acetonitrile Ka-values of 4.6 +/- 0.4 x 10(3) M(-1) and 6.5 +/- 1.2 x 10(3) M(-1), respectively, were calculated. The Ka-values are about four times larger than those obtained with UV/vis spectroscopy in solution, probably due to a higher ionization efficiency of complexed compared to uncomplexed Zn-porphyrin. For the complexation of N-(1-adamantyl)acetamide (8) with beta-cyclodextrin (7), a Ka-value of 3.6 +/- 0.3 x 10(4) M(-1) was obtained, which is in good agreement with that determined by microcalorimetry.

We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single nucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics.