The Unfolded Protein Response in a Pair of Sensory Neurons Promotes Entry of C. elegans into Dauer Diapause

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Neuronal KGB-1 JNK MAPK signaling regulates the dauer developmental decision in response to environmental stress in Caenorhabditis elegans

In response to stressful growth conditions of high population density, food scarcity, and elevated temperature, young larvae of nematode Caenorhabditis elegans can enter a developmentally arrested stage called dauer that is characterized by dramatic anatomic and metabolic remodeling. Genetic analysis of dauer formation of C. elegans has served as an experimental paradigm for the identification and characterization of conserved neuroendocrine signaling pathways. Here, we report the identification and characterization of a conserved c-Jun N-terminal Kinase-like mitogen-activated protein kinase (MAPK) pathway that is required for dauer formation in response to environmental stressors. We observed that loss-of-function mutations in the MLK-1-MEK-1-KGB-1 MAPK pathway suppress dauer entry. A loss-of-function mutation in the VHP-1 MAPK phosphatase, a negative regulator of KGB-1 signaling, results in constitutive dauer formation, which is dependent on the presence of dauer pheromone but independent of diminished food levels or elevated temperatures. Our data suggest that the KGB-1 pathway acts in the sensory neurons, in parallel to established insulin and TGF-β signaling pathways, to transduce the dauer-inducing environmental cues of diminished food levels and elevated temperature.     

Microfluidic conductimetric bioreactor

A microfluidic conductimetric bioreactor has been developed. Enzyme was immobilized in the microfluidic channel on poly-dimethylsiloxane (PDMS) surface via covalent binding method. The detection unit consisted of two gold electrodes and a laboratory-built conductimetric transducer to monitor the increase in the conductivity of the solution due to the change of the charges generated by the enzyme-substrate catalytic reaction. Urea–urease was used as a representative analyte-enzyme system. Under optimum conditions urea could be determined with a detection limit of 0.09 mM and linearity in the range of 0.1–10 mM (r = 0.9944). The immobilized urease on the microchannel chip provided good stability (>30 days of operation time) and good repeatability with an R.S.D. lower than 2.3%. Good agreement was obtained when urea concentrations of human serum samples determined by the microfluidic flow injection conductimetric bioreactor system were compared to those obtained using the Berthelot reaction (P < 0.05). After prolong use the immobilized enzyme could be removed from the PDMS microchannel chip enabling new active enzyme to be immobilized and the chip to be reused.     

Development and application of a real-time capacitive sensor

A real-time capacitive sensor based on a potentiostatic step method was developed. It can display in real-time the evoked current waveform, capacitance and the electrical resistance of elements serially connected to the insulation layer on the electrode as a function of time as well as the ohmic resistance of the insulation layer. These features enable the user to observe the association and dissociation of the affinity binding pairs and to evaluate the insulating property of the electrode surface during measurement. The system allows the setting of potential pulse height, pulse interval, gain, filter, and sampling frequency, enabling the system to be more flexible. The performance of the system was firstly evaluated with equivalent circuits. Under suitable parameter settings it provided good accuracy of both the capacitance and resistance. Using the affinity binding pair of human serum albumin (HSA) and anti human serum albumin (anti-HSA) the measured capacitance change was used for the direct detection of HSA. The developed system provided the same sensitivity as the commercially available potentiostat (P>0.05). The proposed system was then applied to analyse HSA in real urine samples and the results agreed well with the immunoturbidimetric assay (P>0.05). The proposed system can be applied for capacitance measurement to directly detect other target analytes using different affinity binding pairs. Other applications such as kinetics analysis of the interaction between affinity bindings, thickness analysis, and the study of the insulation property of the modified layer are also promising.     

Label-free capacitive DNA sensor using immobilized pyrrolidinyl PNA probe: Effect of the length and terminating head group of the blocking thiols

This paper reports, for the first time, the influence of the length and the terminating head group of blocking thiols on the sensitivity and specificity of a label-free capacitive DNA detection system using immobilized pyrrolidinyl peptide nucleic acid (acpcPNA) probes. A C-terminal lysine-modified acpcPNA was immobilized through four different alkanethiol self-assembled monolayers (SAMs), i.e., 3-mercaptopropionic acid (MPA), thioctic acid (TA), thiourea (TU) and mercaptosuccinic acid (MSA). The hybridization between the acpcPNA probes and the target DNA was directly measured using the capacitive system. Five blocking thiols of various lengths (C=3, 6, 8, 9 and 11), with the -OH terminating head group, i.e., 3-mercapto-1-propanol (3-MPL), 6-mercapto-1-hexanol (6-MHL), 8-mercapto-1-octanol (8-MOL), 9-mercapto-1-nonanol (9-MNL), 11-mercapto-1-undecanol (11-MUL) and another blocking thiol (C=11) with a -CH(3) terminating head group, and 1-dodecanethiol (1-DDT) were investigated. The blocking thiol with the same length as the total spacer of the immobilized acpcPNA gave the highest sensitivity and specificity with the -OH terminating head group providing a slightly better signal than the -CH(3) group. Under the optimized conditions, the immobilized acpcPNA probes provided a wide linear range for DNA detection (1.0×10(-11)-1.0×10(-8)M) with a very low detection limit in the picomolar range. The modified acpcPNA electrode could be reused through at least 58 cycles. The high sensitivity and very low detection limits are potentially useful for the analysis of ultra-trace levels of DNA in samples. Preliminary studies were also performed to see the effect of probe concentration and target length.     

A comparative study of capacitive immunosensors based on self-assembled monolayers formed from thiourea, thioctic acid, and 3-mercaptopropionic acid

A procedure was developed for the covalent coupling of anti-alpha-fetoprotein antibody (anti-AFP) to a gold surface modified with a self-assembled monolayer (SAM) of thiourea (TU). The performance of the SAM-antibody layer was compared to those of similar layers based on thioctic acid (TA) and 3-mercaptopropionic acid (MPA) by using flow injection capacitive immunosensor system. Covalent coupling of anti-AFP on self-assembled thiourea monolayer (SATUM) modified gold electrode can be used to detect alpha-fetoprotein with high efficiency, similar sensitivity, the same linear range (0.01-10 microgl(-1)) and detection limit (10 ngl(-1)) as those obtained from sensors based on self-assembled thioctic acid monolayer (SATAM) and self-assembled 3-mercaptopropionic acid monolayer (SAMPAM). The system is specific for alpha-fetoprotein and can be regenerated and reused up to 48 times. Therefore, self-assembled monolayer using thiourea which is cheaper than thioctic acid and 3-mercaptopropionic acid is a good alternative for biosensor applications when SAMs are used.     

Ultra trace analysis of small molecule by label-free impedimetric immunosensor using multilayer modified electrode

A multilayer electrode modified with a self-assembled thiourea monolayer (SATUM) followed by gold nanoparticles (AuNPs), mercaptosuccinic acid (MSA) and antibody was investigated for the detection of ultra trace amount of a small molecule (chloramphenicol) in an impedimetric system. The formation of the antibody-antigen complex at the electrode surface caused the impedance to increase. Under optimum conditions three modified electrodes were compared the SATUM/AuNPs/MSA electrode provided a wide linear range (0.50-10) × 10?1? M, and a very low determination limit of 1.0 × 10?1? M. This determination limit was much lower than the SATUM/AuNPs electrode, 1.0 × 10?1? M, and SATUM electrode, 4.7 × 10?1? M. The modified electrode provided good selectivity for chloramphenicol detection and can be reused up to 45 times with a relative standard deviation of lower than 4%. When applied to determine chloramphenicol in shrimp samples, the results agreed well with those obtained by the high-performance liquid chromatography coupled with a photo diode array detector (P > 0.05). The developed system can be applied to detect other small molecules using appropriate affinity binding pairs.     

Comparative study of controlled pore glass, silica gel and poraver for the immobilization of urease to determine urea in a flow injection conductimetric biosensor system

This study compared the responses of three enzyme reactors containing urease immobilized on three types of solid support, controlled pore glass (CPG), silica gel and Poraver. The evaluation of each enzyme reactor column was done in a flow injection conductimetric system. When urea in the sample solution passed though the enzyme reactor, urease catalysed the hydrolysis of urea into charged products. A lab-built conductivity meter was used to measure the increase in conductivity of the solution. The responses of the enzyme reactor column with urease immobilized on CPG and silica gel were similar and were much higher than that of Poraver. Both CPG and silica gel reactor columns gave the same limit of detection, 0.5 mM, and the response was still linear up to 150mM. The analysis time was 4-5 min per sample. The enzyme reactor column with urease immobilized on CPG gave a slightly better sensitivity, 4% higher than the reactor with silica gel. The life time of the immobilized urease on CPG and silica gel were more than 310h operation time (used intermittently over 7 months). Good agreement was obtained when urea concentrations of human serum samples determined by the flow injection conductimetric biosensor system was compared to the conventional methods (Fearon and Berthelot reactions). These were statistically shown using the regression line and Wilcoxon signed rank tests. The results showed that the reactor with urease immobilized on silica gel had the same efficiency as the reactor with urease immobilized on CPG.     

Development of low-cost microfluidic systems for lab-on-a-chip biosensor applications

In this work, we develop low-cost microfluidic systems based on polydimethylsiloxane (PDMS) for lab-on-a-chip applications. PDMS microfluidic structures have been fabricated by micromolding, PDMS casting, and plasma bonding processes. The micromolding technique is used to fabricate PDMS slabs with micro-sized grooves, and the complete microchannel is formed by bonding PDMS slab with glass or PDMS substrate. The molding procedure using SU-8 photoresist patterning on silicon wafer, PDMS microchannel fabrication, and PDMS surface treatment using oxygen plasma and TiO₂ coating, are discussed. The various parameters for oxygen plasma treatment including RF power and treatment time are studied in order to obtain conditions for good bonding with the glass substrate. The best condition for plasma treatment is found to be the low RF power (8 W) with 5 min treatment time. In addition, TiO₂ coating with oxygen plasma treatment has been applied to make PDMS surface more hydrophilic to improve aqueous solution compatilbility. The microfluidic channels for various applications, including sample injection cross channel, micropump channel, T and Y sample mixers, PCR thermocyling chamber and channel, capillary electrophoresis flow channel, and conductimetric systems have been fabricated. Finally, a typical application of the PDMS chip in a flow injection conductimetric system for sodium chloride detection has been demonstrated.