Integrated sample-to-detection chip for nucleic acid test assays.

Nucleic acid based diagnostic techniques are routinely used for the detection of infectious agents. Most of these assays rely on nucleic acid extraction platforms for the extraction and purification of nucleic acids and a separate real-time PCR platform for quantitative nucleic acid amplification tests (NATs). Several microfluidic lab on chip (LOC) technologies have been developed, where mechanical and chemical methods are used for the extraction and purification of nucleic acids. Microfluidic technologies have also been effectively utilized for chip based real-time PCR assays. However, there are few examples of microfluidic systems which have successfully integrated these two key processes. In this study, we have implemented an electro-actuation based LOC micro-device that leverages multi-frequency actuation of samples and reagents droplets for chip based nucleic acid extraction and real-time, reverse transcription (RT) PCR (qRT-PCR) amplification from clinical samples. Our prototype micro-device combines chemical lysis with electric field assisted isolation of nucleic acid in a four channel parallel processing scheme. Furthermore, a four channel parallel qRT-PCR amplification and detection assay is integrated to deliver the sample-to-detection NAT chip. The NAT chip combines dielectrophoresis and electrostatic/electrowetting actuation methods with resistive micro-heaters and temperature sensors to perform chip based integrated NATs. The two chip modules have been validated using different panels of clinical samples and their performance compared with standard platforms. This study has established that our integrated NAT chip system has a sensitivity and specificity comparable to that of the standard platforms while providing up to 10 fold reduction in sample/reagent volumes.

Sensitivity and Response of Polyvinyl Alcohol/Tin Oxide Nanocomposite Multilayer Thin Film Sensors.

Nanocrystalline Tin Oxide (SnO2) is Non-Stoichiometric in Nature with Functional Properties Suitable for gas sensing. In this study, SnO2nanoparticles were prepared by the sol-gel technique, which were then characterised using X-ray diffraction. The nanoparticles showed tetragonal structure with an average crystallite size of 18 nm. The stretching and vibration modes of SnO2were confirmed using Fourier transform infrared spectroscopy. The size of SnO2 nanoparticles was determined using particle size analyser, which was found be 60 ± 10 nm on average. The surface morphology of the nanoparticles was investigated using scanning electron microscope, which showed irregular-sized agglomerated SnO2nanostructures. In addition, primary particle size was evaluated using high-resolution transmission electron microscopy, which was found to be 50 nm on average. The polyvinyl alcohol/SnO2 composite thin film was prepared on a glass substrate using spin-coating method. The values of band gap energy and electrical conductance of 13-layer thin film were found to be 2.96 eV and 0.0505 mho, respectively. Sulfur dioxide (SO2) was suitably tailored to verify the sensor response over a concentration range of 10-70 ppm at room temperature. The performance, response, and recovery time of sensors were increased by increasing the layers of the thin film.

Droplet Microfluidic Chip Based Nucleic Acid Amplification and Real-Time Detection of Influenza Viruses.

Miniaturized bio-diagnostic devices have the potential to allow for rapid pathogen screening in clinical patient samples, as a low cost and portable alternative to conventional bench-top equipment. Miniaturization of key bio-diagnostic techniques, such as: nucleic acid detection and quantification, polymerase chain reaction (PCR), DNA fingerprinting, enzyme linked immunosorbent assay (ELISA), results in substantial reduction of reaction volumes (expensive samples/reagents) and shorter reaction times. Droplet microfluidics (DMF) is one of several miniaturized bio-sample handling techniques available for manipulating clinical samples and reagents in microliter (10-6 L) to picoliter (10-12 L) volume regime. Electro-actuation of sample and reagent in the form of droplets in the aforementioned volume regime, using dielectrophoresis (DEP) and/or Electrowetting (EW) are achieved by means of patterned, insulated metal electrodes on one or more substrates. In this work, we have utilized electro-actuation based DMF technology, integrated with suitably tailored resistive micro-heaters and temperature sensors, to achieve chip based real-time, quantitative PCR (qRT-PCR). This qRT-PCR micro-device was utilized to detect and quantify the presence of influenza A and C virus nucleic acids, using in-vitro synthesized viral RNA segments. The experimental analysis of the DMF micro-device confirms its capabilities in qRT-PCR based detection and quantification of pathogen samples, with accuracy levels comparable to established commercial bench-top equipment (PCR efficiency ∼95%). The limit of detection (LOD) of the chip based qRT-PCR technique was estimated to be ∼5 copies of template RNA per PCR reaction.

Higher-order dielectrophoretic effects: levitation at a field null.

Experiments with certain new micro-electrode structures used to achieve passive dielectrophoretic levitation of small particles and biological cells reveal a pronounced size-dependent effect not anticipated by the conventional dipole-based model. The conventional theory fails to predict this size effect because it neglects higher-order moments such as the quadrupole, hexapole, and octupole. These higher-order moments are in fact responsible for the levitation force achieved by azimuthally periodic electrode structures because, in such geometries, the electric field is zero along the axis so that the induced dipole moment must be zero. For example, the planar quadrupole levitates particles passively along the central axis through the interaction of its field with the induced quadrupolar moment of the particle. The size effect reported with this structure is readily explained in terms of this quadrupolar component of the ponderomotive force exerted on the particle.

Dielectrophoretic spectra of single cells determined by feedback-controlled levitation.

In this paper we have utilized the principle of dielectrophoresis (DEP) to develop an apparatus to stably levitate single biological cells using a digital feedback control scheme. Using this apparatus, the positive DEP spectra of both Canola plant protoplast and ligament fibroblast cells have been measured over a wide range of frequencies (1 kHz to 50 MHz) and suspending medium conductivities (11-800 muS/cm). The experimental data thus obtained have been interpreted in terms of a simple spherical cell model. Furthermore, utilizing such a model, we have shown that various cellular parameters of interest can be readily obtained from the measured DEP levitation spectrum. Specifically, the effective membrane capacitance of single cells has been determined. Values of 0.47 +/- 0.03 muF/cm2 for Canola protoplasts and 1.52 +/- 0.26 muF/cm2 for ligament fibroblasts thus obtained are consistent with those determined by other existing electrical methods.

Dipole interactions in electrofusion. Contributions of membrane potential and effective dipole interaction pressures.

The contributions of pulse-induced dipole-dipole interaction to the total pressure acting normal to the membranes of closely positioned pronase treated human erythrocytes during electrofusion was calculated. The total pressure was modeled as the sum of pressures arising from membrane potential and dipole-dipole attraction opposed by interbilayer repulsion. The dipole-dipole interaction was derived from the experimentally obtained cell polarizability. The threshold electric field amplitude necessary for fusion of pronase-treated human erythrocytes was experimentally obtained at various combinations of pulse duration, frequency, and the conductivity of the external medium. The theoretical values of the critical electric field amplitude compared favorably to the experimentally obtained threshold field amplitudes. Fusion by dc pulses may be primarily attributed to attainment of sufficiently high membrane potentials. However, with decreasing external conductivity and increasing sinusoidal pulse frequency (100 kHz-2.5 MHz), the induced dipole-dipole interactions provide the principal driving force for membrane failure leading to fusion.

A novel dielectrophoresis-based device for the selective retention of viable cells in cell culture media.

Cost-effective production of biopharmaceuticals on a large scale can be carried out by perfusion cultures of mammalian cells. One problem with this mode of operation for submerged free-cell cultures is the requirement for an efficient cell separation device located in the effluent stream. The present work investigates the potential for the development of a novel dielectrophoresis-based cell separator, capable of providing selective retention of viable cells in cell culture media, which are highly conductive. Predictions of the dielectrophoretic (DEP) response in culture media were first obtained through a series of DEP-levitation experiments. Subsequently, a prototype microelectrode "filter" was microfabricated and tested with C174 myeloma cell suspensions of density 1 x 10(6) cells/mL. The optimum frequency range for selective retention of viable cells was found in the range 5-15 MHz. A maximum separation efficiency of 98% was achieved at 10 MHz, with an applied peak-to-peak voltage of 30 V (maximum field strength of 10(5) V/m) and a flow rate of 30 mL/h which corresponds to a superficial velocity of 5.23 cm/h through the DEP-filter channels.

Dual-frequency dielectrophoretic levitation of Canola protoplasts.

A novel dual-frequency excitation technique is introduced which permits investigation of the low-frequency dispersion of Canola plant protoplasts using feedback-controlled dielectrophoretic levitation. The upper and intermediate frequency spectra obtained using the new technique are generally consistent with previous work. However, below some cross-over frequency f(OL), the protoplasts exhibit an apparent positive dielectrophoretic response that is not predicted by conventional theory. This cross-over frequency is linearly related to suspension conductivity, virtually independent of the suspension pH, and inversely proportional to the square of the cell radius. Examination of the complex Clausius-Mossotti polarization coefficient reveals that the observed positive dielectrophoretic response can not be accounted for in terms of Maxwell-Wagner polarization associated with a conventional layered model for the protoplast. The failure of straightforward enhancements to the protoplast model in explaining the low frequency behavior may indicate the presence of an electrophoretic contribution to the net observable force on the particle. To account for such fluid mechanical effects, it will be necessary to modify the existing dielectrophoretic force formulation.