Microextraction Methods And Applications Based On Microfluidic Droplet Technology

The miniaturization development of modern analytical science puts forward higher requirements for sample pretreatment technology,and microfluidic technology provides an important platform for the miniaturization of analytical science.As an important branch of microfluidic technology,the droplet microfluidic technology is a new technology that has been rapidly developed in recent years to manipulate droplets of picoliter to nano liter volume.It has the advantages of less reagent and sample consumption,fast mass and heat transfer,less sample diffusion and cross-contamination.These features make the droplet microfluidic system very suitable for pretreatment of ultra-micro samples during pretreatment analysis.and is very suitable for the pre-treatment and analysis of ultra-micro samples.The present work explored the microextraction method based on the droplet microfluidic technology,and established the nanoliter droplet-droplet microfluidic microextraction(DDMME)method and micro solid phase microextraction(μSPME)method.The two established methods were combined with MALDI-TOF MS and the LC-MS,respectively,for the analysis and determination of two drug metabolites in nanoliter biological samples with complex matrix(HepG2 cell droplets and human liver microsomal droplets).In Chapter 1,the existing microextraction techniques,including liquid-phase microextraction(LPME),microfluidic chip based microextraction,and solid-phase microextraction(SPME)were described.In addition,the droplet microfluidic technology,the analytical techniques for droplet detection,and their main applications in the biochemical analysis were classified and summarized.In Chapter 2,we developed a droplet-droplet microfluidic microextraction(DDMME)system by combining droplet-based microfluidics,robotic liquid handling and LPME techniques for achieving fast and efficient microextraction between both microvolume sample and extractant in the nanoliter range.Two microextraction modes,droplet-in-droplet microfluidic microextraction(DID-MME)and droplet-on-droplet microfluidic microextraction(DOD-MME)were developed according to different solubility properties of the extractants.In order to optimize two microextraction modes,100 nL of RGB droplets(5.0×10-4 mol/L)were used as the sample droplets,and 10 nL of chloroform and n-hexanol droplets were used as the extractant droplets in the DID-MME and DOD-MME modes,respectively.The factors affecting the microextraction performance were investigated,including the extraction time,recovery method of the extractant droplet,static and dynamic extraction modes,and cross-contamination through oil.Compared with the conventional liquid-liquid extraction(LLE)methods,the reagent and sample consumption in DDMME could be reduced at least 5 orders of magnitude;the extraction time was reduced from tens of minutes to several minutes;the procedures in DDMME were automated and flexible.This system was rapid,flexible and could be coupled with a variety of chromatographic and spectroscopic methods.In Chapter 3,we combined the DID-MME method with matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)for the determination of drug metabolites in individual nanoliter cell droplets,providing an effective platform for the analysis of ultra-miniature samples in the nanoliter range.The anticancer drug irinotecan(CPT-11)was used as a model drug and 7-ethyl-10-hydroxy-20-camptothecin(SN-38),an active metabolite of CPT-11,in individual nanoliter HepG2 cell droplets was quantitatively determined.The extraction conditions including extraction temperature,extractant droplet volume,extraction solvent,and extraction time were optimized.Under the optimum conditions,a linear relationship was obtained for SN-38 in the range of 4-100 ng/mL(R2=0.984).The limits of detection(LOD)and quantitation(LOQ)of SN-38 were 2.2 ng/mL and 4.5 ng/mL,respectively.The recovery was 84.5%-94.8%,and the RSD value was 6.1%(n=6).The method was simple,rapid,and could meet the requirements for the analysis and determination of the drug metabolite SN-38 in the actual sample HepG2 cell droplets.In Chapter 4,we developed a micro solid-phase microextraction(μSPME)method by combining droplet microfluidics,robotic liquid handling,and solid-phase microextraction(SPME)techniques.The μSPME probe prepared in this method used quartz fiber with a tip of 2 μm as the substrate,and polyacrylonitrile(PAN)and C18 microspheres were used as the particle binder and adsorptive particles,respectively.In the experiments,we optimized the experimental conditions for the modification of the PAN/C18 coating.We selected 800 nL of RGB droplets(5.0×10-5 mol/L)as model sample droplets.Potential factors affecting the extraction process,such as the extraction time,static and dynamic extraction modes,desorption solvent and desorption time were studied in detail.In addition,the measurement RSDs of single and multiple μSPME probes were 0.5%(n=6)and 2.2%(n=6),respectively.Therefore,the method is fast in operation,low in cost,repeatable,biocompatible and is especially suitable for pre-treatment analysis of ultra-micro biochemical samples.In Chapter 5,we established a μSPME/LC-MS system based on droplet microfluidics for the determination of target analytes in nanoliter-scale samples with complex matrices.In this system,the tip of the μSPME probe was directly inserted into the inlet of the capillary column to complete the desorption,which minimized the sample loss caused by desorption and injection,etc.This system was used to determine acetaminophen,a metabolite of phenacetin,in nanoliterscale human liver microsomal droplets.In the experiment,the experimental conditions including extraction time,desorption solvent,and desorption time were investigated.Under the optimum conditions,a linear relationship was obtained for acetaminophen in the range of 20500 μmol/L(R2=0.991).The limits of detection(LOD)and quantitation(LOQ)of acetaminophen were 9.6 μmol/L and 19.1 μmol/L,respectively.Under optimized extraction conditions,the RSD value of acetaminophen was 8.3%(n=6)Therefore,the newly establishedμSPME/LC-MS system has the advantages of flexible operation,minimum sample loss,strong separation ability,and good repeatability,and has the potential to simultaneously analyze and determine multiple components in ultramicro samples with complex matrices simultaneously.