| Sinec the global outbreak of COVID-19,infectious diseases caused by pathogens are once again in the spotlight.Nucleic acid amplification detection plays an important role in the diagnosis of diseases as it could identify nucleic acids specific sequences of the pathegon within high sensitive ablitiy.By integrating nulceic acids detection in a microfluidic chip,the nucleic acids diagnosis process can be integrated on a small,closed microfluidon-on-chip laboratory,enabling non-specialists to perform point-ofcare tesing(POCT)nucleic acid testing in environments other than traditional molecular diagnostic laboratories.As the first progress of molecular diagnosis,nucleic acids extraction process directly determines the type of sample to be processed and the quality of template.The majority methods of nucleic acid extraction mainly include phenol-chloroform extraction as liquid phase extraction method,and solid phase extraction method based on silica membrane or magnetic beads.The main problems are as follows:(1)these nucleic acid extraction process was mainly based on solid medium with high specific surface area(magnetic microspheres or silica membrane),which is complicated and time-consuming generally;(2)the material did not take advantage of the intrinsic characteristics of substrance in nucleic acid extraction progress,instead of only as a crude intergration of the traditional nucleic acid extraction method,combined with redundant valve or pump.As a result,the chip design would be more complex and costly.Point at these problems,this paper focuses on how to use the intrinsic characteristics of microfluidic chips to achieve efficient nucleic acid extraction,simplify the process of nucleic acid detection on chips,and reduce the complexity of chip design.Therefore,this paper innovatively proposes a new method for pathogen nucleic acid detection based on SIMPLE Chip,with the following characteristics:(1)(Surface area-to-volume Irrespective)The chip channel itself is used as the enrichment medium for nucleic acid extraction,instead of the traditional high surface area-tovolume magnetic such as beads or silica membranes.(2)chitosan was Modified in the channel of the chip to optimize the nucleic acid binding liquid system to realize the Point-of-care and Low-cost-based Extrcation of pathogen nucleic acid.3)After enrichment of nucleic acid by chitosan modified chip,nucleic acid elution is not required,which simplifies the process of pathogen nucleic acid detection,and is suitable for detection of nucleic acid which is low concentration.The main contents of this paper are as follows:(1)Research on the key process of SIMPLE Chip preparation based on low-cost polymer materials was carried out,which laid the chip foundation for the efficient enrichment and in situ amplification detection of pathogen nucleic acids.Firstly,a sandwich structure of SIMPLE chip is designed.The main structure of the chip consisted of "glass substrate-PDMS film-PMMA substrance".On the main substrance of the chip,four chambers with low surface area was assembled for nucleic acid enrichment,in situ amplification and detection.The laser parameters on the cutting effect of the SIMPLE chip was study,and the results showed that the optimized parameters as:defocus distance was 1 mm,the cutting speed was 10%,the laser frequency was 5000 Hz,and the laser pulse width was 30 ?s,the effect of unit structure was studied.The structure characteristics of each unit(aperture/edge roughness)of the SIMPLE chip were accroded with the requirements of chip assembly and bonding.Then,the hot pressing assisted oxygen plasma bonding method was investigated,and the fast bonding of "glass substrate-PDMS film-PMMA "was realized,the production cycle was less than 8 min for a single simple chip in average.Fluid shock and cooling test results showed that the SIMPLE Chip could toleranced the rapid inflow and outflow of fluid over 10,000 times the volume of the chamber.The SIMPLE Chip also could toleranced rapid increase and cooling in the PCR progress with 45 amplification cycles without any reagent leakage.The results provided a laid the foundation for in situ amplification of pathogen nucleic acid detection.In order to realized the efficient enrichment of pathogen nucleic acid in the chmaber with low specific surface area,the process of modifying SIMPLE chip with chitosan(a positively charged material)was explored by using the principle of the opposites attract of charge.Characterization analysis including hydrophilic angle measurement,AFM,FTIR and XPS proved that SIMPLE Chip was successfully modified by chitosan,and the modified SIMPLE chip was still characterized by low specific surface area.After that,the modified SIMPLE Chip had no amplification inhibition in PCR progeress was also demostrated.(2)Nnucleic acid extraction and in situ amplification detection method based on SIMPLE Chip was reseached,and efficient enrichment and in situ amplification detection of pathogen nucleic acid on SIMPLE Chip were realized.First of all,a serials of experiment method were used to reseached the feature of nucleic acid enrichment ability of SIMPLE chipunder different experimental conditions(enrichment time,ion environment,etc.),thus excellent enrichment performance on pathogen nucleic acid was determined.The adsorption kinetics results showed that SIMPLE Chip could achieve the maximum adsorption capacity of nucleic acid in 2 min,the adsorption capacity of nucleic acid was 105-130 ng/cm2,and the enrichment efficiency was about 75%,indicating that SIMPLE Chip has the advantages of rapid enrichment of nucleic acid and high load.Whatmore,the SIMPLE Chip could be directly amplified in situ without nucleic acid elution after purification progress.The detection sensitivity is 4000-8000 times higher than that of SIMPLE Chip without modified chitosan,and 410 times higher than that of traditional commercial nucleic acid extraction kits.(3)Asmart phone pathogen nucleic acid detection system matching with SIMPLE Chip was designed and devoloped,which solved the problem that conventional pathogen nucleic acid detection methods relayed on the professional laboratories and technical personnel mostly.The POCT device for pathogen Nucleic acid detection integrated the temperature control unit,fluorescence detection unit,SIMPLE Chip,and other necessities required for nucleic acid detection.The weight of the detection system was about 1.5kg,the rising and cooling rates were 6.5?/s and 5.59?/s,respectively.The temperature uniformity was less than 0.1?,and 45 amplification cycles could be completed within 1 h.TracePro simulation showed a peak irradiance of around 85%in the central region,means good fluorescence uniformity on the device,thus a consistent detection platform for the SIMPLE chip was confirmed.Finally,the SIMPLE Chip and smart phone based nucleic acid extraction and in situ amplification detection method were evaluated using Zika pseudovirus.The experimental results showed that the sensitivity of SIMPLE Chip was 10 TU/mL,better than the commercial nucleic acid extraction kit and amplification instrument.And the performance even could be better for low concentration samples(10-100 TU/mL).In conclusion,in view of the requirement of pathogen nucleic acid detection,this paper intruded the research on the crucial process of SIMPLE Chip preparation based on low-cost polymer materials,and developed the nucleic acid extraction-in situ amplification reagent system for SIMPLE chip.In addition,we developed a smart phone pathogen nucleic acid detection system with The SIMPLE Chip,realizing a highly sensitive detection method for Zika virus in saliva,providing a new method and new ideas for the development of point-of-care testing(POCT)technology. |
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