Using dielectrophoretic spectra to identify and separate viable yeast cells.

Immotile yeast cells were transiently moved in nonuniform sinusoidal electric fields using multiple pairs of micro-parallel cylindrical electrodes equipped with a sequential signal generator (SSG) to analyze cell viability at a clinical scale for the brewery/fermentation industry. Living yeast cells of Saccharomyces cerevisiae during the exponential-stationary phase, with a cell density of 1.15 × 105 cells mL-1 were suspended in sucrose medium. The conductivity (σs) was adjusted to 0.01 S m-1 with added KCl. Cells exposed in electric field strengths ranging from 32.89 to 40.98 kV m-1, exhibited positive dielectrophoresis (pDEP) with the lower critical frequencies (LCF) at 85.72 ± 3.59 kHz. The optimized value of LCF was shifted upwards to 780.00 ± 83.67 kHz when σswas increased to 0.10 S m-1. Dielectrophoretic and LCF spectra (translational speed of cells vs. electric field frequencies) of yeast suspensions during positive dielectrophoresis were analyzed in terms of the dielectric properties of the cell membrane and cytoplasm which reflect yeast cell viability and metabolic health status. The dielectrophoretic collection yield of cells using positive dielectrophoresis was reported on the monitor of sequential signal generator software to evaluate the number of living and dead cells through a real-time image processing analyzer. The spectra of both positive dielectrophoresis of the living and dead cells had distinguishable dielectric properties. The conductivity of the yeast cytoplasm (σc) of the dead cells was significantly less (≈ ≤ 0.05 S m-1) than that of the living yeast cells which typically had a cytoplasmic conductivity of ≈ 0.2 S m-1. This difference between viable and non-viable cells is sufficient for cell separation procedures. KEY POINTS: • Dielectrophoresis can be used to separate viable and non-viable yeast cells, • Cellular dielectric properties can be derived from the analysis of their dielectric spectra, • Cytoplasmic conductivity of viable cells is ≈ 0.2 S m-1 while that of non-viable cells ≈ ≤ 0.05 S m-1.

Real-time multianalyte biosensors based on interference-free multichannel monolithic quartz crystal microbalance.

In this work, we design, fabricate and characterize a new interference-free multichannel monolithic quartz crystal microbalance (MQCM) platform for bio-sensing applications. Firstly, interference due to thickness-shear vibration mode coupling between channels in MQCM array is effectively suppressed by interposing a polydimethylsiloxane wall between adjacent QCM electrodes on a quartz substrate to form inverted-mesa-like structure. In addition, the electrical coupling due to the electrical impedance of solution is diminished by extending the flow path between them with an extended-design flow channel. The electrical testing results show that individual QCM signal is unaffected by those of adjacent channels under liquid loading, signifying the achievement of interference-free MQCM. The MQCM is applied for multi-analyte biosensing of IgG and HSA. The anti-IgG and anti-HSA are separately immobilized on two adjacent QCM electrodes, which are subsequently blocked with BSA to avoid unspecific binding. The MQCM biosensors are tested with single- and double-analyte solutions under continuous flow of buffer. The IgG and HSA QCM sensors only show frequency shift responses to their corresponding analytes and there are very small cross frequency shifts due to remnant unspecific binding. Moreover, MQCM sensors show approximately linear frequency shift response with analyte concentration. Therefore, the developed MQCM platform is promising for real-time interference-free label-free detection and quantification of multiple bio-analytes.

Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample.

Pathogenic Vibrio cholerae produces a cholera toxin which is the cause of a severe diarrheal disease called "Cholera". Available detection methods, including standard bacteriological test and immuno-based detection, are specific to the suspected pathogenic V. cholerae O1 and O139, but they are not specific to the cholera toxin producible strain. This work combined the polymerase chain reaction (PCR) of cholera toxin gene, ctxA gene, and microcantilever-based DNA sensor to improve the sensitivity and specificity of detection. Gold coated microcantilever, 250 µm long and 50 µm wide, with an embedded polysilicon wire acting as a piezoresistive material was modified by a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) for immobilization of specific DNA probe via avidin layer on the surface. The avidin and 5' biotinylated single-stranded DNA (ssDNA) probe concentrations were optimized for the immobilization at 50 µg/mL and 1 µM, respectively. The hybridization between ssDNA probe on this DNA sensor and target DNA creates nanomechanical bending and resistance change of piezoresistive material inside the beam. This microcantilever-based DNA sensor offers a detection sensitivity of 3.25 pg or 14 nM of DNA template for ctxA gene detection. The lowest number of V. cholerae O1 in food sample with and without the enrichment process that the polymerase chain reaction (PCR) for ctxA gene combined with this DNA sensor can detect is 0.835 and 835 cells/g, respectively. This detection sensitivity is 10 times higher than that of the conventional PCR method.

Enhancement of DNA hybridization under acoustic streaming with three-piezoelectric-transducer system.

Recently, we have demonstrated that DNA hybridization using acoustic streaming induced by two piezoelectric transducers provides higher DNA hybridization efficiency than the conventional method. In this work, we refine acoustic streaming system for DNA hybridization by inserting an additional piezoelectric transducer and redesigning the locations of the transducers. The Comsol® Multiphysics was used to design and simulate the velocity field generated by the piezoelectric agitation. The simulated velocity vector followed a spiral vortex flow field with an average direction outward from the center of the transducers. These vortices caused the lower signal intensity in the middle of the microarray for the two-piezoelectric disk design. On the contrary, the problem almost disappeared in the three-piezoelectric-disk system. The optimum condition for controlling the piezoelectric was obtained from the dye experiments with different activation settings for the transducers. The best setting was to activate the side disks and middle disk alternatively with 1 second activating time and 3 second non-activating time for both sets of transducers. DNA hybridization using microarrays for the malaria parasite Plasmodium falciparum from the optimized process yielded a three-fold enhancement of the signal compared to the conventional method. Moreover, a greater number of spots passed quality control in the optimized device, which could greatly improve biological interpretation of DNA hybridization data.

A review of monolithic multichannel quartz crystal microbalance: a review.

Monolithic multichannel quartz crystal microbalance (MQCM) is an emerging technology for advanced sensing and measurement applications. In this report, a comprehensive review of MQCM technology is presented. Firstly, basic MQCM's design, simulation and characterization with emphasis on acoustic interference are described. Next, various MQCM schemes to minimize interference and enhance sensitivity of conventional MQCM devices based on modification of quartz substrate structure are digested. These include mesa, convex and x-axis inversion structures. Three important MQCM sensing platforms and their application areas are then discussed. These comprise MQCM as a static multichannel detector, series MQCM as a multichannel detector for the flow injection analysis and multi-frequency QCM for multi-sensitivity/multi-dynamic range detection. Finally, potential MQCM applications including electronic noses, bio-sensor arrays, and photocatatalytic measurement are illustrated and prospective MQCM applications including electronic tongues and electrochemical measurement are suggested.

DNA hybridization enhancement using piezoelectric microagitation through a liquid coupling medium.

In conventional DNA microarray hybridization, delivery of target cDNAs to surface-bounded probes depends solely on diffusion, which is notoriously slow, and thus typically requires 6-20 h to complete. In this study, piezoelectric microagitation through a liquid coupling medium is employed to enhance DNA hybridization efficiency and the results are compared with the standard static hybridization method. DNA hybridization was performed in a sealed aluminium chamber containing DNA microarray glass chip, coupling medium and piezoelectric transducers. 3×SSC (Saline Sodium Citrate) was used as a coupling medium to prevent overheating of the piezoelectric transducers and to effectively transmit ultrasonic wave to the glass chip. Flow visualization using fluidic dye and velocimetry (PTV) technique was applied to observe fluid transport in the hybridization chamber. It was revealed that the dye solution was homogeneously distributed within 10 min under dynamic agitation while it took over 1 h to reach the same level of homogeneity in static condition. Plasmodium falciparum DNA microarrays and total RNA extracted from parasite cells were used as a model for DNA microarray experiments. It was found that the required hybridization time may be substantially reduced from 16 h to 4 h by the use of dynamic hybridization scheme. With the same hybridization time of 16 h, dynamic hybridization resulted in higher fluorescent signals of ∼33% and ∼24% compared to static hybridization in Cy3 and Cy5 channels, respectively. Additionally, good/effective spots, some of which were not formed by static method, were enhanced and distributed more uniformly over the microarray. Therefore, the developed dynamic hybridization with integrated piezoelectric microagitation platform is highly promising for DNA analysis in molecular biology and medical applications.

Cardiac troponin T detection using polymers coated quartz crystal microbalance as a cost-effective immunosensor.

Cardiac troponin T (cTnT) detection has been the focus of increased interest due to its role in myocardial infarction diagnosis. In this study, we report a relatively low coat technique to detect cTnT using a quartz crystal microbalance (QCM) sensor. A sensitive detection is achieved by introducing a QCM surface with a carboxylic polyvinyl chloride immobilization layer. The surface morphologies of this polymer film under varied deposition thickness have been investigated by field emission scanning electron microscopy and atomic force microscopy. A cTnT detection result from a modified QCM surface can be obtained within a short response time by a direct detection of the immunoreaction and a direct conversion of mass accumulation into a frequency shift, representing a measurable electrical signal. The relationship between the cTnT concentration and the response current from a QCM sensor shows detectability at the concentration of cTnT as low as 5 ng/ml.

Flow injection based microfluidic device with carbon nanotube electrode for rapid salbutamol detection.

A microfabicated flow injection device has been developed for in-channel electrochemical detection (ECD) of a beta-agonist, namely salbutamol. The microfluidic system consists of PDMS (polydimethylsiloxane) microchannel and electrochemical electrodes formed on glass substrate. The carbon nanotube (CNT) on gold layer as working electrode, silver as reference electrode and platinum as auxiliary electrode were deposited on a glass substrate. Silver, platinum, gold and stainless steel catalyst layers were coated by DC-sputtering. CNTs were then grown on the glass substance by thermal chemical vapor deposition (CVD) with gravity effect and water-assisted etching. 100-microm-deep and 500-microm-wide PDMS microchannels fabricated by SU-8 molding and casting were then bonded on glass substrate by oxygen plasma treatment. Flow injection and ECD of salbutamol was performed with the amperometric detection mode for in-channel detection of salbutamol. The influences of flow rate, injection volume, and detection potential on the response of current signal were optimized. Analytical characteristics, such as sensitivity, repeatability and dynamic range have been evaluated. Fast and highly sensitive detection of salbutamol have been achieved. Thus, the proposed combination of the efficient CNT electrode and miniaturized lab-on-a-chip is a powerful platform for beta-agonists detection.