[Genetic diagnosis for a family without exonic deletions and duplications of dystrophin gene].

OBJECTIVE: To conduct genetic diagnosis for a family in which no exonic deletions and duplications of the dystrophin gene were detected. METHODS: Potential exonic deletions and duplications of the dystrophin gene were initially analyzed with using multiplex ligation-dependent probe amplification (MLPA). Subsequently, all of the 79 exons of the dystrophin gene of the proband and a pregnant woman from the family were analyzed with PCR amplification and DNA sequencing. Following identification of the causative mutation, prenatal diagnosis was provided. RESULTS: MLPA analysis had detected no exonic deletions and duplications of the dystrophin gene. Sequence analysis has identified a C>T mutation on the 22nd nucleotide position of the 70th exon of the dystrophin gene (c.10108 C>T), which has replaced the codon CGA to a stop codon (TGA). The patient's mother and sister were both heterozygous for the same mutation. Upon prenatal diagnosis, the fetus was found to be positive for the Y chromosome sex-determining gene (SRY) and has carried above mutation. The result of short tandem repeat linkage analysis also confirmed that the fetus has inherited the mutant X chromosome. CONCLUSION: The causative mutation of the dystrophin gene has been discovered in an affected family, which has enabled prenatal diagnosis of the disease.

Development of an enzyme-linked immunosorbent assay (ELISA) to measure the level of tyrosine hydroxylase protein in brain tissue from Parkinson's disease models.

Tyrosine hydroxylase (TH) catalyses the rate-limiting step in the biosynthesis of catecholamines. TH expression is regulated in a tissue-specific manner during neuronal development and differentiation. Because of its key regulatory role in central and peripheral catecholamine synthesis, TH is associated with the pathogenesis of several neurological and psychiatric diseases, including Parkinson's disease, dystonia, schizophrenia, affective disorders, and cardiovascular diseases. Therefore, developing a quantitative method to monitor the changes in TH expression in disease models could facilitate the identification and characterisation of neuromodulatory and neuroprotective therapeutic agents. The present report describes the generation and characterisation of a new set of monoclonal TH antibodies and the development of a novel sandwich ELISA for the quantitative detection of the TH protein in rodent brain tissue. This ELISA exhibits excellent reproducibility and good linearity in the analysis of complex brain tissue lysates. The cross-validation of the TH ELISA using semi-quantitative TH Western blot methods and HPLC measurement of dopamine levels suggests that the new TH ELISA is sufficiently sensitive to detect small-to-moderate region-specific differences, developmental changes, and Parkinson's disease-related changes in TH expression in rodent brains. This new TH ELISA also offers greater flexibility than conventional HPLC-based dopamine assays because the optimal tissue lysis buffer used for the detection of TH in brain tissue is also compatible with the analysis of other proteins associated with Parkinson's disease, such as α-synuclein, suggesting that this TH ELISA could be used in a multiplexed format.

Thick-film textile-based amperometric sensors and biosensors.

The incorporation of amperometric sensors into clothing through direct screen-printing onto the textile substrate is described. Particular attention is given to electrochemical sensors printed directly on the elastic waist of underwear that offers tight direct contact with the skin. The textile-based printed carbon electrodes have a well-defined appearance with relatively smooth conductor edges and no apparent defects or cracks. Convenient voltammetric and chronoamperometric measurements of 0-3 mM ferrocyanide, 0-25 mM hydrogen peroxide, and 0-100 muM NADH have been documented. The favorable electrochemical behavior is maintained under folding or stretching stress, relevant to the deformation of clothing. The electrochemical performance and tolerance to mechanical stress are influenced by the physical characteristics of the textile substrate. The results indicate the potential of textile-based screen-printed amperometric sensors for future healthcare, sport or military applications. Such future applications would benefit from tailoring the ink composition and printing conditions to meet the specific requirements of the textile substrate.

Flexible thick-film glucose biosensor: influence of mechanical bending on the performance.

The influence of the bending-induced mechanical stress of flexible Nafion/GOx/carbon screen-printed electrodes (SPEs) upon the performance of such glucose biosensors has been examined. Surprisingly, such flexible enzyme/polymer-SPEs operate well following a severe bending-induced mechanical stress (including a 180 degrees pinch), and actually display a substantial sensitivity enhancement following their mechanical bending. The bending-induced sensitivity enhancement is observed only for the amperometric detection of the glucose substrate but not for measurements of hydrogen peroxide, catechol or ferrocyanide at coated or bare SPEs. These (and additional) data indicate that the bending effect is associated primarily with changes in the biocatalytic activity. Such sensitivity enhancement is more pronounced at elevated glucose levels, reflecting the bending-induced changes in the biocatalytic reaction. Factors affecting the bending-induced changes in the performance are examined. While our data clearly indicate that flexible enzyme/polymer-SPEs can tolerate a severe mechanical stress and hold promise as wearable glucose biosensors, delivering the sample to the active sensor surface remains the major challenge for such continuous health monitoring.

Effects of electric potential treatment of a chromium hexacyanoferrate modified biosensor based on PQQ-dependent glucose dehydrogenase.

A novel potential treatment technique applied to a glucose biosensor that is based on pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and chromium hexacyanoferrate (CrHCF) incorporated into a platinum (Pt) electrode was demonstrated. CrHCF, serving as a mediator, was electrochemically deposited on the Pt electrode as ascertained by CV, SEM, FTIR and XPS measurements. The potential treatment of CrHCF, which converts Fe(II) to Fe(III), enables the glucose detection. The amperometric measurement linearity of the biosensor was up to 20 mM (R = 0.9923), and the detection sensitivity was 199.94 nA/mM per cm(2). More importantly, this biosensor remained stable for >270 days.

Thermally Stable Improved First-Generation Glucose Biosensors based on Nafion/Glucose-Oxidase Modified Heated Electrodes.

We illustrate how the use of heated electrodes enhances the performance of glucose biosensors based on amperometric detection of the glucose-oxidase generated hydrogen peroxide. Nafion is shown to be an excellent matrix to protect glucose oxidase from thermal inactivation during the heating pulses. The influence of the electrode temperature upon the amperometric response is examined. Temperature pulse amperometry (TPA) has been used to obtain convenient peak-shaped analytical signals. Surprisingly, up to 67.5 °C, the activity of Nafion-entrapped glucose oxidase is greatly enhanced (24 -fold) by accelerated kinetics rather than decreased by thermal inactivation. Amperometric signals even at elevated temperatures are stable upon prolonged operation involving repetitive measurements. The linear calibration range is significantly extended.

A miniaturized glucose biosensor for in vitro and in vivo studies.

A miniaturized wireless glucose biosensor has been developed to perform in vitro and in vivo studies. It consists of an external control subsystem and an implant sensing subsystem. The implant subsystem consists of a micro-processor, which coordinates circuitries of radio frequency, power regulator, command demodulator, glucose sensing trigger and signal read-out. Except for a set of sensing electrodes, the micro-processor, the circuitries and a receiving coil were hermetically sealed with polydimethylsiloxane. The electrode set is a substrate of silicon oxide coated with platinum, which includes a working electrode and a reference electrode. Glucose oxidase was immobilized on the surface of the working electrode. The implant subsystem bi-directionally communicates with the external subsystem via radio frequency technologies. The external subsystem wirelessly supplies electricity to power the implant, issues commands to the implant to perform tasks, receives the glucose responses detected by the electrode, and relays the response signals to a computer through a RS-232 connection. Studies of in vitro and in vivo were performed to evaluate the biosensor. The linear response of the biosensor is up to 15 mM of glucose in vitro. The results of in vivo study show significant glucose variations measured from the interstitial tissue fluid of a diabetes rat in fasting and non-fasting periods.

Using MPTMS as permselective membranes of biosensors.

A sol-gel material of (3-mercaptopropyl) trimethoxysilane (MPTMS) is proposed to function as permselective membranes of biosensors. Permselectivity of MPTMS and Nafion was compared by studying their anti-interferent ability. Membrane porosity of MPTMS and Nafion was first confirmed via voltammetric responses in ferrocynite/ferricynite solution. In the comparison studies, membranes prepared with 20% MPTMS in phosphate buffer solution (PBS) and 1% Nafion in 2-propanol (IPA) were used as coating materials on the surface of two platinum (Pt) electrodes. These electrodes were used to electrochemically measure the response currents of ascorbic acid, uric acid, and acetaminophen. The results indicate that the MPTMS-based electrode produced much less response currents from the interference species compared to that of the Nafion-based electrode. This denotes that the anti-interferent ability ofMPTMS is superior to that of Nafion. A platinum working electrode containing glucose oxidase (GOx) immobilized by poly-aniline (PA) and then modified by MPTMS was developed and evaluated. The results show that the optimum applied potential for the glucose biosensor is 0.4 V. This operational potential not only inhibits the response currents from ascorbic acid, uric acid, and acetaminophen but also produces rather high signals for glucose.

Chromium hexacyanoferrate modified biosensor based on PQQ-dependent glucose dehydrogenase.

A Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase based platinum electrode was developed to detect glucose. Chromium hexacyanoferrate was modified onto this electrode to serve as an electron transfer mediator between PQQ and the platinum electrode. This biosensor showed the optimal response of glucose measurements at pH 7 and an operation potential of +0.4 V (vs. Ag/AgCl). More importantly the lifespan of the biosensor has been last for 47 days without significant enzyme activity degradation.