1. Field of the Invention
The present invention generally relates to a detection device and method for charged molecular targets, and more particularly to a membrane based lateral-flow device and method to enhance the detection signal for charged molecular targets, to enhance the detection sensitivity.
2. Description of the Prior Art
Immunochromatography is an easy and fast detection method, and users, even when not well-trained, can determine test results without the need for or use of any extra instruments. Because of its convenient operation and relatively quick readout capability, immunochromatography is wildly applied in the fields of environment testing, food testing, bio-chemical weapons, clinical medicine, etc. According to the capillarity of different membranes with different density and specific immunity between targets and marked reagents, the color of the marked reagents can be read to indicate whether the targets exist, and even to show the concentration of the targets.
The above-mentioned immunochromatography characteristics are based upon antigen-antibody immunoassay considerations. Similarly, the binding specificity between DNA-ligand and anti-ligand bodies is employed to detect nucleic acid targets by the hybridization reaction in a rapid and convenient manner.
Nitrocellulose is usually used for the material of the lateral-flow membrane. Since the nitrocellulose has strong affinity to the molecular targets, the capillarity-induced movements of some molecular targets are trapped in the membrane. Therefore, not all targets flow to the test line and show its distinguished color, and the threshold of the detection limit is not ideal. Only molecular targets with high concentration in samples suit this traditional detection approach. In this regard, it is necessary to provide an improvement method for reducing the remains of the molecular targets on the membrane to increase the sensitivity.
In view of the foregoing omission of the prior art, one object of the present invention is to provide a membrane based lateral-flow detection device for charged molecular targets with a sensitive detection limit. This detection device includes a membrane based lateral-flow detection assembly and a direct current source.
Another object of the present invention is to enhance the movement of the charged molecular targets by the direct current source, whereby the detection sensitivity is therefore increased.
According to the foregoing objects, the present invention provides a membrane based lateral-flow method and device. This membrane based lateral-flow device includes a membrane based lateral-flow detection assembly and a direct current source. With the adding of direct current, the movement of the charged molecular targets is enhanced, the remains of the molecular targets on the membrane pad are reduced, and the detection limit is increased.
A detailed description of the present invention will be provided in connection with the following embodiments, which are not intended to limit the scope of the present invention and which can be adapted to other applications. While the drawings are illustrated in detail, it is appreciated that the quantity of the disclosed components may be greater or fewer than that disclosed, except where the amount of such components is expressly restricted.
In accordance with an embodiment of the present invention,
When the electricity of the charged molecular targets is positive, the first electrode should be chosen to be an anode, and the second electrode should be chosen to be a cathode. On the contrary, when the electricity of the charged molecular targets is negative, the first electrode should be chosen to be a cathode, and the second electrode should be chosen to be an anode. The direct current source can come from the batteries, or provided by the inverter/transformer converting from the alternating current.
With comparative reference to
The foregoing conjugate pad includes a label, a labeled antibody, and a labeled secondary antibody or a labeled antigen, wherein the label includes fixed-radioactive substance (such as I125, H3, C14 and P32), enzyme, fluorescence material, dye, carbon black, colloid gold and latex.
The foregoing molecular targets are not limited by the embodiment of the present invention. Those charged samples suit the detection device revealed by the present invention, such as Deoxyribonucleic acid (DNA), RiboNucleic Acid (RNA), protein, amino acid, bio-molecules, medicine, drugs and specialty chemicals.
The avian influenza is chosen to be the target, and the DNA targets are H5 sequence in A/Singapore/1/57 (H2N2). First, single-stranded DNA product is amplified by asymmetrical Polymerase Chain Reaction (PCR) and dig-labeled. After purification, targets react with the anti-ligand antibody which is labeled with a color label (e.g., colloid gold). Then, targets flow to the test zone, and hybridization reaction is performed to show the color of colloid gold. The detection result is designed to be visually read out by gold-sol to identify distinct sorts of Avian Influenza.
Viruses such as avian influenza have RNA genomes that can be converted into complementary DNA (cDNA) by an enzyme called reverse transcriptase. cDNA is a more convenient way to work with the coding sequence than mRNA because RNA is very easily degraded by omnipresent RNases. This is the main reason why cDNA is sequenced rather than mRNA. Likewise, investigators conducting DNA microarrays often convert the mRNA into cDNA in order to produce their probes. The cDNA of avian influenza is then amplified by asymmetrical PCR, and analyzed by agarose gel electrophoresis.
Since the segment of DNA is 358 mer in length and might move slow (e.g., slowly) down (e.g., downwardly) due to its bulky structure, a direct current voltage is added to enhance the detection signal.
A complementary probe cH5 is included in the test line 32 of the present invention. The control line possesses Rabbit anti-mouse IgG. The conjugate pad 20 possesses mouse anti digoxigenin labeled nano-gold which reacts with the dig part in the targets cH5. The sample pad 10 contains dig-labeled H5 PCR product.
The probe cH5 in the test line 32 will hybridize with the PCR product, and digoxigenin of PCR product will capture gold-conjugated mouse anti digoxigenin to show the color of nano-gold. Besides, Rabbit anti-mouse IgG in the control line 34 will react with gold conjugated mouse anti digoxigenin to show the color of nano-gold.
Generally, the result of agarose gel-electrophoresis is very important to bio-molecule detection limit. Since the principle of the membrane based lateral-flow detection device for the charged molecular targets in the present invention is something similar to the agarose gel-electrophoresis, it is necessary to execute an agarose gel-electrophoresis experiment with different H5 concentration for comparison. 2% Agarose gel and 30 ml 0.5× TBE buffer solution are used for 35 minutes gel-electrophoresis, then Agarose gel is soaked in Ethidium bromide (EtBr) for 20 minutes. Finally, UV lamp or UV lightbox is used to visualize DNA in the gel.
As shown in
In step 403, a direct current source which includes a first electrode and a second electrode is provided, wherein the electricity of the first electrode is different from that of the second electrode. The first electrode is located between the conjugate pad and the test line, and the second electrode is located between the control line and the absorbent pad. The direct current source provides an electromotive force to drive the charged molecular targets to flow from the first electrode to the second electrode, and the electricity of the charged molecular targets is different from that of the second electrode.
Although specific embodiments have been illustrated and described, it will be appreciated by those skilled in the art that various modifications may be made without departing from the scope of the present invention, which is intended to be limited solely by the appended claims.
Number | Date | Country | Kind |
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097151595 | Dec 2008 | TW | national |