Disposable plastic microreactors for genomic analyses.

We show the design, development and assessment of disposable, biocompatible, fully plastic microreactors, which are demonstrated to be highly efficient for genomic analyses, such as amplification of DNA, quantitative analyses in real time, multiplex PCR (both in terms of efficiency and selectivity), as compared to conventional laboratory equipment for PCR. The plastic microreactors can easily be coupled to reusable hardware, enabling heating/cooling processes and, in the case of qPCR applications, the real-time detection of the signal from a suitable fluorescent reporter present in the reaction mixture during the analysis. The low cost production of these polymeric microreactors, along with their applicability to a wide range of biochemical targets, may open new perspectives towards practical applications of biochips for point of care diagnostics.

Chronic pain as expression of neural substrates. Issues from the neuronal dynamics and mutual relations.

The Thalamo-Cortical somatosensory loop shows important synaptic re-organization in cases of chronic pain. Animal models exhibit severe functional distortions, potentially related to the anatomic rearrangements. Connectivity and information theoretic measurement represent important tools to quantify the functional disarrays. We performed electrophysiological experiments with multisite, multielectrode simultaneous recordings in the Thalamus and in the Somatosensory Cortex. The recurrent anomalies in the analytic estimates induce to hypothesize a potential neurodynamical explanation of the sensory context.

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 1).

The performances and the stability of a novel subcutaneous glucose monitoring system have been evaluated. GlucoDay (A. Menarini I.F.R. S.r.l, Florence Italy) is a portable instrument provided with a micro-pump and a biosensor coupled to a microdialysis system capable of recording the subcutaneous glucose level every 3 min. Long and short term stability of the biosensor are discussed and the results of some critical in vitro and in vivo (on rabbits) experiments are reported. A linear response up to 30 mM has been found for in vivo glucose concentration. The sensitivity referred to blood glucose is better than 0.1 mM and the zero current is typically below the equivalent of 0.1 mM. In the accuracy study a mean bias of 2.7 mg/dl and a correlation coefficient equal to 0.9697 have been found. At room temperature, an excellent membrane stability assures good performances up to 6 months from the first use.

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 2).

The aim of this study was to evaluate the reproducibility, the accuracy and the reliability of a continuous subcutaneous glucose measuring system. The GlucoDay system (A. Menarini I.F.R. S.r.l.-Florence, Italy) is a portable instrument provided with a micro-pump and a biosensor, coupled to a microdialysis system (see part 1). This instrument has demonstrated high reliability coupled with a low degree of invasivity. The profiles of glucose monitoring allow to achieve an excellent knowledge of the real variation of glucose in diabetic patients. The reproducibility study showed a bias lower than 10% between instruments. The accuracy study showed a difference from the reference method lower than 15%.

Enzymatic reactions for the determination of sugars in food samples using the differential pH technique.

All ATP coupled reactions, when performed at neutral or moderately alkaline pH, produce an acidification of the reaction mixture. The detection of small pH changes--0.1 mpH (1 mpH = 10(-3) pH)--in a constant buffering capacity solution makes it possible to quantify, over a wide concentration range (1-1500 mmol L(-1)), various analytes with very high precision and accuracy. Glucose, fructose, glycerol and gluconic acid can be analysed in less than 1 min with a single step reaction. Wine samples were analysed using the hexokinase reaction for glucose + fructose (sugars undergoing fermentation) and compared against an established method, showing excellent performance over the whole range of concentrations (R = 0.9994). Increased sensitivity in some applications can be obtained by cycling reactions, e.g. a kinase reaction followed by a phosphatase reaction. in a one step analysis, as required for lactulose assay in milk, a useful indicator of heat treatment damage. A sensitivity well below 0.1 mmol L(-1) in the original milk sample has been demonstrated.

Mixed-mode fluorescent DNA sequencing.

Fluorescence-based, automated DNA sequences represent one of the major advances in recent molecular biology. Two main technologies have been developed in this field: the single-label/four-lane system and the four-label/one-lane system. The following present the use of single-label-sequencing chemistry, which resembles traditional radioactive DNA sequencing, using the four-label system ABI 373A that expands its flexibility and obtains data that are immediately interpretable without software manipulation. This method has been named mixed-mode fluorescent DNA sequencing. Here we show one of its possible applications in molecular genetic analysis.

Introduction of magnetic beads in diagnosis: a simple and rapid method to detect mutations of beta globin gene, directly amplified from blood.

In this communication we present the results obtained by the use of magnetic beads in diagnosis, for the identification of genetic variants at the molecular level by sequencing, in comparison with the more laborious method of the production of ssDNA with asymmetric PCR. We compared the two techniques studying variants of beta globin gene: Hb Abruzzo [beta 143 (H21) His -> Arg] and Hb D Los Angeles [beta 121 (GH4) Glu -> Gln].

Clinical utility of fractionating erythrocytes into "Percoll" density gradients.

Two rapid methods for fractionating the RBC into five or nine layers of increasing density are reported. These procedures have been used to monitor the decline of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activity during the process of red cell aging in normal subjects and in beta-thal carriers, to study transfused patients with G6PD and pyruvate kinase (PK) deficiency and to test the effects of inositol hexaphosphate (IHP) encapsulation on RBC subpopulations.