Research On The Detection Technology Of Biological Macromolecules Based On Micro-droplet Array

Microfluidic technology is a technology that controls fluids on the micrometer scale.It can concentrate multiple steps of sample detection on a small chip,and separate the sample to multiple independent reaction units through multiple channels to achieve high efficiency and high throughput sample detection.It has the characteristics of miniaturization,automation,less consumption and little pollution.As an important branch of microfluidic technology research,droplet microfluidic technology combines the characteristics of droplet technology and traditional microfluidic technology to avoid the difficulty of continuous fluid fusion and the complexity of chip structure.At the same time,it has the characteristics of flexible manipulation,fast analysis speed and good monodispersity.It has built a brand new platform for biological and medical research,especially suitable for the analysis and detection of biological macromolecules such as DNA and proteins.In this paper,a droplet biological reaction platform device based on microfluidic printing technology is developed,which can generate addressable and controllable droplet arrays with high throughput.This device has studied the detection technology of different biological macromolecules in tiny volumes by dispersing biological macromolecules in the generated droplet array.The specific research contents are as follows:(1)A biological reaction platform based on microfluidic impact printing technology was developed,which includes a biological droplet generation device,a biological droplet reaction temperature control device,and a biological droplet detection device.In the research on droplet generation,a droplet generation method and a multi-volume printing method are proposed,and the printing effect is demonstrated and the uniformity of droplet generation is verified;The droplet reaction conditions were explored from the application parameters of droplet reaction prevention evaporation,droplet contact angle of different materials and different substrate materials;The fluorescence and the colorimetric are proposed to complete the detection of macromolecules in the droplet array.It has the characteristics of high integration,less reagent consumption and wide application range.(2)A droplet digital PCR technology based on microfluidic impact printing(MIP-ddPCR)is proposed.The microfluidic impact printing technique was used to generate a nanoliter droplet array containing DNA on a surface-modified quartz substrate,which was amplified by a flat-plate thermal cycler and counted by a scanning fluorescence microscope to complete the absolute quantification of rare genes.It is a low-cost and pollution-free gene detection technology under laboratory conditions.In addition,multi-volume droplet digital PCR technology can be realized by in-situ printing method,thereby expanding the detection concentration range.In the experiment,we verified the measurement accuracy of the MIP-ddPCR technology on four orders of magnitude by detecting a series of concentration gradients of the GAPDH gene,and applied it to detect the expression level of targeted genes in colon cancer samples,showing the potential of this method in clinical samples.(3)A method for rapid droplet generation for biomacromolecule detection was developed.This method uses a reusable double-sided adhesive mask and plasma treatment to form a hydrophilic and hydrophobic surface on the PMMA material,which can form a large-scale droplet array.This method can well control the volume of droplets by adjusting the size of the wettability pattern on the substrate.In the experiment,we verified the biocompatibility of the plasma-treated PMMA substrate.At the same time,combined with microfluidic impact printing technology,the detection of different concentrations of human serum albumin and the induced expression of green fluorescent protein in E.coli were completed.This proves the application potential of the droplet array as a more convenient and economical biochemical analysis and detection platform.In summary,we use microfluidic impact printing technology to develop a biological reaction platform that can generate addressable,volume controllable microdroplet arrays,suitable for the detection of a variety of biological macromolecules.In specific applications,we have implemented a droplet digital PCR method based on microfluidic impact printing technology.Only a microfluidic printing device,a common PCR amplifier and fluorescence microscope in the laboratory can complete the absolute quantification of genes,especially suitable for the detection of rare biological targets under laboratory conditions.At the same time,combination of microfluidic impact printing technology and self-separating droplet array,quantitative detection of biological samples such as human serum albumin with different concentrations has been completed.It has the characteristics of less reagent consumption,fast detection speed and wide detection range,which has great application potential as a new biochemical analysis and detection platform.