Study Of Cotton Thread Based Rapid Bioassay Method

In this paper, a novel cotton thread-based rapid bioassay device was explored for lung cancer biomarkers (squamous cell carcinoma antigen and human ferritin) detection. In this paper, a novel molecular beacon was developed for rapid detection of a human genetic disease, hereditary tyrosinemia type I related DNA sequences. The ability of the novel molecular beacon for discriminating the single base mismatched DNA related to a human genetic disease, hereditary tyrosinemia type I, was improved. The main pionts of this thesis are summarized as follow:(1) Study of disposable dry-reagent cotton thread-based point-of-care diagnosis device for SCCAIn this paper, author reported here by using cotton thread-based rapid diagnosis device for low-cost, sensitive and rapid detection of a lung cancer related biomarker, squamous cell carcinoma antigen (SCCA). A pair of monoclonal antibodies are detection antibody and capture antibody. The capture antibody bound tightly to the surface of the cotton thread through a combination of electrostatic and hydrophobic interactions. An enhancement protocol was employed by using two kinds of gold nanoparticle labels for SCCA test which greatly improved the sensitivity of the device. Two kinds of gold nanoparticle labels were Au NP-dAb-bDNA conjugates and Au NP-SA conjugates. For quantitative measurements, the optical intensity of the red bands was read using the scanner combined with "ImageJ" software. The thread based immunoassay device was capable of measuring 5 ng/mL SCCA in 20 minutes which meet the requirement for clinical diagnosis.(2) Study of a novel adenosine-based molecular beacon probe for nucleic acid rapid detection in cotton thread deviceIn this paper, author used cotton thread as substrate to develop a novel DNA detection device for low-cost, sensitive and rapid detection of a human genetic disease, hereditary tyrosinemia type I related DNA sequences.the DNA probe has a thiol group and a biotin group on each end. A novel adenosine based molecular beacon (ABMB) probe modified on gold nanoparticle was used as reporter probe. In the presence of coralyne, a small molecule which can react with adenosines, the ABMB would form a hairpin structure just like traditional molecular beacon used extensively. In the presence of target DNA sequences, the hairpin structure of ABMB modified on gold nanoparticles will be opened and the biotin group modified at one end of the DNA probes will be released and react with the streptavidin immobilized on the test zone of the cotton thread. A red band can be observed with naked eyes as a qualitative test mean. Quantification detection was read using the scanner combined with "Image J" software. The response of the thread based DNA test device is linear over the range of 2.5-100 nM complementary DNA. The ability of our developed device for discriminating the single base mismatched DNA related to a human genetic disease, hereditary tyrosinemia type I, was improved comparing with previous report. It’s worth mentioning that the whole assay procedure for DNA test is performed under room temperature which simplified the assay procedures greatly.(3) Study of a novel room temperature nucleic acid detection method based on immobilization of adenosine-based molecular beaconIn this paper, two kinds of adenosine-based molecular beacon probes with twelve adenosines bases at each end (A12-MBI-A12 and A12-MB2-A12), and their complementary target DNA, non-complementary target DNA and single base mismatched DNAs were used as models to demonstrate the proof-of-concept. Under optimized conditions, the detection limit was estimated to be 50 pM based on S/N=3. The ability of our developed biosensor for discriminating the single base mismatched DNA related to a human genetic disease, hereditary tyrosinemia type I, was improved undoubtedly once A12-MB2-A12 was used as capture probe comparing with previous report. It’s worth to mention that the whole assay procedures were conducted under room temperature.(4) Study of novel gold nanoparticle trimer reporter probe combined with point-of-care diagnosis cotton thread based immunoassay device for human ferritin testIn this paper, author reported here on the use of cotton thread-based combined with novel gold nanoparticle trimer reporter probe for low-cost, sensitive and rapid detection of a lung cancer related biomarker, human ferritin. The capture antibody bound tightly to the surface of the cotton thread through a combination of electrostatic and hydrophobic interactions. A model system comprising human ferritin as an analyte and a pair of monoclonal antibodies was used to demonstrate the proof-of-concept on the dry-reagent cotton thread based immunoassay device. On the test zone formed a sandwich Au NP-dAb-human ferritin-cAb complexes. Results indicated that using of novel gold nanoparticle trimer reporter probe greatly improved in terms of sensitivity comparing with traditional gold nanoparticle reporter probe on the cotton thread based immunoassay device. The cotton thread based immunoassay device was capable of measuring 10 ng/mL human ferritin in 20 minutes which meet the requirement for clinical diagnosis.