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.