Magneto-capillary valve for integrated purification and enrichment of nucleic acids and proteins.

We describe the magneto-capillary valve (MCV) technology, a flexible approach for integrated biological sample preparation within the concept of stationary microfluidics. Rather than moving liquids in a microfluidic device, discrete units of liquid are present at fixed positions in the device and magnetic particles are actuated between the fluids. The MCV concept is characterized by the use of two planar surfaces at a capillary mutual distance, with specific features to confine the fluids by capillary forces, and the use of a gas or a phase-change material separating the stationary aqueous liquids. We have studied the physics of magneto-capillary valving by quantifying the magnetic force as a function of time and position, which reveals the balance of magnetic, capillary and frictional forces in the system. By purification experiments with a fluorescent tracer we have measured the amount of co-transported liquid, which is a key parameter for efficient purification. To demonstrate the versatility of the technology, several MCV device architectures were tested in a series of biological assays, showing the purification and enrichment of nucleic acids and proteins. Target recovery comparable to non-miniaturized commercial kits was observed for the extraction of DNA from human cells in buffer, using a device architecture with patterned air valves. Experiments using an enrichment module and patterned air valves demonstrate a 40-fold effective enrichment of DNA in buffer. DNA was also successfully purified from blood plasma using paraffin phase-change valves. Finally, the enrichment of a protein biomarker (prostate-specific antigen) using geometrical air valves resulted in a 7-fold increase of detection signal. The MCV technology is versatile, offers extensive freedom for the design of fully integrated systems, and is expected to be manufacturable in a cost-effective way. We conclude that the MCV technology can become an important enabling technology for point-of-care systems with sample in-result out performance.

Quantifying the composition of human skin for glucose sensor development.

BACKGROUND: Glucose is heterogeneously distributed within human skin. In order to develop a glucose measurement method for human skin, both a good quantification of the different compartments of human skin and an understanding of glucose transport processes are essential. This study focused on the composition of human skin. In addition, the extent to which intersubject variability in skin composition alters glucose dynamics in human skin was investigated. METHODS: To quantify the composition of the three layers of human skin-epidermis, dermis, and adipose tissue-cell and blood vessel volumes were calculated from skin biopsies. These results were combined with data from the literature. The composition was applied as input for a previously developed computational model that calculates spatiotemporal glucose dynamics in human skin. The model was used to predict the physiological effects of intersubject variability in skin composition on glucose profiles in human skin. RESULTS: According to the model, the lag time of glucose dynamics in the epidermis was sensitive to variation in the volumes of interstitial fluid, cells, and blood of all layers. Data showed most variation/uncertainty in the volume composition of the adipose tissue. This variability mainly influences the dynamics in the adipose tissue. CONCLUSIONS: This study identified the intersubject variability in human skin composition. The study shows that this variability has significant influence on the glucose dynamics in human skin. In addition, it was determined which volumes are most critical for the quantification and interpretation of measurements in the different layers.

DNA computing using single-molecule hybridization detection.

DNA computing aims at using nucleic acids for computing. Since micromolar DNA solutions can act as billions of parallel nanoprocessors, DNA computers can in theory solve optimization problems that require vast search spaces. However, the actual parallelism currently being achieved is at least a hundred million-fold lower than the number of DNA molecules used. This is due to the quantity of DNA molecules of one species that is required to produce a detectable output to the computations. In order to miniaturize the computation and considerably reduce the amount of DNA needed, we have combined DNA computing with single-molecule detection. Reliable hybridization detection was achieved at the level of single DNA molecules with fluorescence cross-correlation spectroscopy. To illustrate the use of this approach, we implemented a DNA-based computation and solved a 4-variable 4-clause instance of the computationally hard Satisfiability (SAT) problem.

The effect of exchange of bacteriopheophytin a with plant pheophytin a on charge separation in Y(M210)W mutant reaction centers of Rhodobacter sphaeroides at low temperature.

The bacteriopheophytin a molecules at the H(A) and H(B) binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of H(A)(-), presumably by raising the free energy level of the P(+)B(A)(-) state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll B(A). Exchange of the bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of H(A), inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P(+)B(B)(-) state, where B(B) is the accessory bacteriochlorophyll in the "inactive" B-branch of the cofactors. The B(B) molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Q(y) region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P(+)B(B)(-) state and absorption properties of the B(B) bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the B(B) binding pocket.