Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support

BACKGROUND: Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization. RESULTS: Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray. CONCLUSION: This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes

A multiplexed immunoassay system based upon reciprocating centrifugal microfluidics

A novel, centrifugal disk-based micro-total analysis system (mu TAS) for low cost and high throughput semi-automated immunoassay processing was developed. A key innovation in the disposable immunoassay disk design is in a fluidic structure that enables very efficient micro-mixing based on a reciprocating mechanism in which centrifugal acceleration acting upon a liquid element first generates and stores pneumatic energy that is then released by a reduction of the centrifugal acceleration, resulting in a reversal of direction of flow of the liquid. Through an alternating sequence of high and low centrifugal acceleration, the system reciprocates the flow of liquid within the disk to maximize incubation/hybridization efficiency between antibodies and antigen macromolecules during the incubation/hybridization stage of the assay. The described reciprocating mechanism results in a reduction in processing time and reagent consumption by one order of magnitude.open121

Serial siphon valving for centrifugal microfluidic platforms

Today, the focus in microfluidic platforms for diagnostics is on the integration of several analysis steps toward sample-to-answer systems. One of the main challenges to integration is the requirement for serial valving to allow the sequential release of fluids in a temporally and spatially controlled manner. The advantages offered by centrifugal microfluidic platforms make them excellent candidates for integration of biological analysis steps, yet they are limited by the lack of robust serial valving technologies. This is especially true for the majority of centrifugal microfluidic devices that rely on hydrophilic surfaces, where few passive serial valving techniques function reliably. Building on the useful functionality of centrifugal microfluidic siphoning previously shown, a novel serial siphon valve is introduced that relies on multiple, inline siphons to provide for a better controlled, sequential release of fluids. The introduction of this novel concept is followed by an analytical analysis of the device. Proof-of-concept is also demonstrated, and examples are provided to illustrate the range of functionality of the serial siphon valve. The serial siphon is shown to be robust and reproducible, with variability caused by the dependence on contact angle, rotation velocity, and fluidic properties (viz., surface tension) significantly reduced compared to current microfluidic, centrifugal serial valving technologies

Serial siphon valving for centrifugal microfluidic platforms

Today, the focus in microfluidic platforms for diagnostics is on the integration of several analysis steps toward sample-to-answer systems. One of the main challenges to integration is the requirement for serial valving to allow the sequential release of fluids in a temporally and spatially controlled manner. The advantages offered by centrifugal microfluidic platforms make them excellent candidates for integration of biological analysis steps, yet they are limited by the lack of robust serial valving technologies. This is especially true for the majority of centrifugal microfluidic devices that rely on hydrophilic surfaces, where few passive serial valving techniques function reliably. Building on the useful functionality of centrifugal microfluidic siphoning previously shown, a novel serial siphon valve is introduced that relies on multiple, inline siphons to provide for a better controlled, sequential release of fluids. The introduction of this novel concept is followed by an analytical analysis of the device. Proof-of-concept is also demonstrated, and examples are provided to illustrate the range of functionality of the serial siphon valve. The serial siphon is shown to be robust and reproducible, with variability caused by the dependence on contact angle, rotation velocity, and fluidic properties (viz., surface tension) significantly reduced compared to current microfluidic, centrifugal serial valving technologies

Internal Control for Nucleic Acid Testing Based on the Use of Purified Bacillus atrophaeus subsp. globigii Spores▿

Commonly used internal controls (ICs) to monitor the efficiency of nucleic acid testing (NAT) assays do not allow verification of nucleic acid extraction efficiency. Since microbial cells are often difficult to lyse, it is important to ensure that nucleic acids are efficiently extracted from any target organism. For this purpose, we developed a cellular IC based on the use of nonpathogenic Bacillus spores. Purified Bacillus atrophaeus subsp. globigii (referred to hereafter as simply B. atrophaeus) spores were added to vaginal and anal samples, which were then subjected to rapid DNA extraction and subsequent PCR amplification. The proof of concept of this cellular IC was made through the use of both manual and automated DNA extraction methods, using vaginal or anal samples spiked with B. atrophaeus spores, combined with a multiplex real-time PCR assay for the specific detection of group B streptococci (GBS) and B. atrophaeus. The performance of the cellular IC was compared to that of a standard IC plasmid added to PCRs. Approximately 500 B. atrophaeus spores per PCR was found to be optimal since this did not interfere significantly with GBS detection for either DNA extraction method and yielded reproducible amplification and/or detection of B. atrophaeus genomic DNA serving as an IC template. Performance of the cellular IC was comparable to that of the standard IC. This novel IC system using nonpathogenic and hard-to-lyse B. atrophaeus spores allowed validation of both the DNA extraction procedure and the amplification and detection process. Use of a spore-based control also provides a universal control for microbial cell lysis

Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays

In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes γ-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics▿

Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations