Limits...
Development of chemiluminescent lateral flow assay for the detection of nucleic acids.

Wang Y, Fill C, Nugen SR - Biosensors (Basel) (2012)

Bottom Line: Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety.On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level.The limit of detection was determined to be 0.5 fmols of nucleic acid target.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, University of Massachusetts, 102 Holdsworth Way, Amherst, MA 01003, USA. yuhongw@foodsci.umass.edu.

ABSTRACT
Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target.

No MeSH data available.


Related in: MedlinePlus

A holder device was machined in plastic in order for the test strips to be inserted into a plate reader. (a) The test strips were placed into the holder and then (b) the cover was placed over the test strips. The entire device had the same measurements as a 96-well plate, and the test and control lines corresponded to wells to allow for reading by a microtiter plate reader.
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biosensors-02-00032-f002: A holder device was machined in plastic in order for the test strips to be inserted into a plate reader. (a) The test strips were placed into the holder and then (b) the cover was placed over the test strips. The entire device had the same measurements as a 96-well plate, and the test and control lines corresponded to wells to allow for reading by a microtiter plate reader.

Mentions: The assay ran as follows: 100 μL of sample solution were first applied onto the sample pad and allowed to migrate towards the absorbent pad. Ten minutes after the sample solution was applied, 100 μL of the 1:1 mixture of Super SigerSignal West Pico Luminol/Enhancer Solution and Supersignal West Pico Stable Peroxide Solution and H2O2 (Peirce Biotechnology, Rockford, IL, USA) were applied onto the sample pad. The intensities of chemiluminescent signals generated on the test and control lines were either quantified in a mitrotiter plate reader (Biotek Winooski, VT, USA) or it was imaged with a CCD imaging station (Kodak, Rochester, NY, USA). In order for the test strips to be read on a microtiter plate reader a device was fabricated to fit into a tray of the reader. The holder device was machined in Delrin™ using a CO2 laser (Epilog Laser, Golden, CO) and designed so that the test lines and control lines corresponded to a well location (Figure 2).


Development of chemiluminescent lateral flow assay for the detection of nucleic acids.

Wang Y, Fill C, Nugen SR - Biosensors (Basel) (2012)

A holder device was machined in plastic in order for the test strips to be inserted into a plate reader. (a) The test strips were placed into the holder and then (b) the cover was placed over the test strips. The entire device had the same measurements as a 96-well plate, and the test and control lines corresponded to wells to allow for reading by a microtiter plate reader.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263543&req=5

biosensors-02-00032-f002: A holder device was machined in plastic in order for the test strips to be inserted into a plate reader. (a) The test strips were placed into the holder and then (b) the cover was placed over the test strips. The entire device had the same measurements as a 96-well plate, and the test and control lines corresponded to wells to allow for reading by a microtiter plate reader.
Mentions: The assay ran as follows: 100 μL of sample solution were first applied onto the sample pad and allowed to migrate towards the absorbent pad. Ten minutes after the sample solution was applied, 100 μL of the 1:1 mixture of Super SigerSignal West Pico Luminol/Enhancer Solution and Supersignal West Pico Stable Peroxide Solution and H2O2 (Peirce Biotechnology, Rockford, IL, USA) were applied onto the sample pad. The intensities of chemiluminescent signals generated on the test and control lines were either quantified in a mitrotiter plate reader (Biotek Winooski, VT, USA) or it was imaged with a CCD imaging station (Kodak, Rochester, NY, USA). In order for the test strips to be read on a microtiter plate reader a device was fabricated to fit into a tray of the reader. The holder device was machined in Delrin™ using a CO2 laser (Epilog Laser, Golden, CO) and designed so that the test lines and control lines corresponded to a well location (Figure 2).

Bottom Line: Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety.On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level.The limit of detection was determined to be 0.5 fmols of nucleic acid target.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, University of Massachusetts, 102 Holdsworth Way, Amherst, MA 01003, USA. yuhongw@foodsci.umass.edu.

ABSTRACT
Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target.

No MeSH data available.


Related in: MedlinePlus