一种基于RepRap开源3D打印平台的低成本、自动化核酸提取系统

A Low-cost,Automated Nucleic Acid Extrac⁃tion System Converted from the Open-Source RepRap 3D Printer

自动化核酸提取技术对于核酸扩增和高通量测序等分子检测应用至关重要。当前,大部分商用自动化核酸提取仪主要以磁分离技术为主,采用高度集成的封闭式结构设计方案,成本高,且不利于在资源有限的环境下使用。此外,核酸样本处理功能模块一旦固定,便很难根据需要对其功能更新和拓展。为了应对上述挑战,该文在基于“快速复制原型机(RepRap)”开源项目的低成本3D打印平台上,设计并构建了一套桌面式8通道自动化核酸提取系统。采用RepRap开源设计,系统模块功能的设计更快速、灵活,可编程,可以根据需要及时进行迭代更新,缩短了系统的研制和测试周期。在开发的系统中,加热、机械运动和8通道磁分离模块功能可与开源3D打印硬件无缝集成。其中,将8通道磁分离模块直接替换3D打印平台的热挤出机,通过G-code编程,可实现自动化核酸提取的高精度定位和机械运动控制。此外,该系统为自动化核酸提取开发了专用实验方案和控制软件。其中,三轴运动平台和核酸提取模块分别由上位机和模块驱动电路进行有效控制;加热模块由4个专门设计的铝制加热底座和3D打印机原有的加热板组成。加热底座通过与96深孔板适配,使深孔板贴合在加热板上,可实现较高的热转换效率。该研究以λDNA为标准核酸样品,对系统的核酸提取纯度、提取效率(一致性和稳定性)进行分析,验证了系统的提取性能。结果显示,该系统对高浓度样品的提纯性能和效率优于低浓度样品。该系统还实现了大肠杆菌细胞质粒DNA的自动提取,进一步展示了其对于真实细胞样本纯化的有效性。该文研制的核酸提取系统有望为在实验室之外的资源有限环境中进行现场快速分子检测(POCT)提供一种经济有效的手段。

Automatic nucleic acid extraction is essential for nucleic acid detection such as DNA amplification and high-throughput sequencing.Currently,most commercial nucleic acid extractors are mainly based on magnetic separa⁃tion technology,adopting a highly integrated structural design or a closed solution,which is too costly and not condu⁃cive to use in resource-limited areas.In addition,it is difficult to update the functionality of the instruments once the functional module is determined.To address these challenges,a desktop automated nucleic acid extraction system was built in this work,which was converted from a low-cost 3D printer based on the Self-Replicating Rapid Prototyp⁃ing(RepRap)open-source project.With the RepRap open-source design,the module function of the system could be flexibly designed and programmable,thus allowing updates as needed,which significantly shortened the manufactur⁃ing and testing cycle of the system.In the developed system,the modular function of the heating,mechanical motion and multi-channel magnetic separation could be fully integrated with the open-source 3D printing hardware.an 8-chan⁃nel magnetic separation based nucleic acid extraction module was designed,which could be assembled onto the origi⁃nal 3-axis motion platform by replacing the 3D printer extruder.The motion path planning required for automatic ex⁃traction was realized by G-code programming.The experimental protocols and control software were developed for the automated nucleic acid extraction in this work.The 3-axis motion platform and nucleic acid extraction module could be effectively controlled by the host computer and the module drive circuit,respectively.The heating module consisted of 4 custom-made aluminum heating bases and the heating plate on the 3D printer.The heating bases were adapted to a 96 deep-well plate,so that the deep-well plate is well attached to the heating block for high heating efficiency.By us⁃ing theλDNA as the standard nucleic acid samples,the extraction performance of the system had been verified by evaluating extraction purity,and efficiency including consistency and stability.From the results,the system showed a better extraction purity and efficiency for the samples with high concentration than that with low concentration.Addi⁃tionally,the 8-channel extraction experiment for the plasmid DNA from the cultured E.coli cells had been success⁃fully carried out on the automated system in this study.This validation further demonstrated that the automated system can be utilized for extracting nucleic acids from real cell samples.Overall,the nucleic acid extraction system devel⁃oped in this study is expected to provide a cost-effective means for rapid point-of-care molecular testing in resourcelimited environments outside the laboratory.