title: Chitosan-Modified Filter Paper for Nucleic Acid Extraction and "in Situ PCR" on a Thermoplastic Microchip.
abstract: Plastic microfluidic devices with embedded chitosan-modified Fusion 5 filter paper (unmodified one purchased from GE Healthcare) have been successfully developed for DNA extraction and concentration, utilizing two different mechanisms for DNA capture: the physical entanglement of long-chain DNA molecules with the fiber matrix of the filter paper and the electrostatic adsorption of DNA to the chitosan-modified filter fibers. This new method not only provided a high DNA extraction efficiency at a pH of 5 by synergistically combining these two capture mechanisms together, but also resisted the elution of DNA from filters at a pH > 8 due to the entanglement of DNA with fibers. As a result, PCR buffers can be directly loaded into the extraction chamber for "in situ PCR", in which the captured DNA were used for downstream analysis without any loss. We demonstrated that the capture efficiencies of a 3-mm-diameter filter disc in a microchip were 98% and 95% for K562 human genomic DNA and bacteriophage λ-DNA, respectively. The washes with DI water, PCR mixture, and TE buffer cannot elute the captured DNA. In addition, the filter disc can enrich 62% of λ-DNA from a diluted sample (0.05 ng/μL), providing a concentration factor more than 30-fold. Finally, a microdevice with a simple two-chamber structure was developed for on-chip cell lysis, DNA extraction, and 15-plex short tandem repeat amplification from blood. This DNA extraction coupled with "in situ PCR" has great potential to be utilized in fully integrated microsystems for rapid, near-patient nucleic acid testing.
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University , Beijing 100084, China.
authors.forename: Wupeng
authors.initials: W
authors.lastname: Gan
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University , Beijing 100084, China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Technology Department of Chaoyang Sub-bureau, Beijing Public Security Bureau , Beijing 100102, China.
authors.forename: Junping
authors.initials: J
authors.lastname: Han
authors.affiliation: Key Laboratory of Forensic Genetics, Beijing Engineering Research Center of Crime Scene Evidence Examination, National Engineering Laboratory for Crime Scene Evidence Examination, Institute of Forensic Science , Beijing 100038, China.
authors.forename: Cai-Xia
authors.initials: CX
authors.lastname: Li
authors.affiliation: Key Laboratory of Forensic Genetics, Beijing Engineering Research Center of Crime Scene Evidence Examination, National Engineering Laboratory for Crime Scene Evidence Examination, Institute of Forensic Science , Beijing 100038, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University , Beijing 100084, China.
authors.forename: Peng
authors.identifier: 0000-0003-3000-3168
authors.initials: P
authors.lastname: Liu
mesh_terms: D048271:Chitosan / Q000737:chemistry*; D004247:DNA / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D005374:Filtration; D064368:Healthy Volunteers; D006801:Humans; D046210:Microfluidic Analytical Techniques; D010209:Paper; D016133:Polymerase Chain Reaction; D013696:Temperature
chemical_list: D004247:DNA; D048271:Chitosan