Droplet Manipulation And Sample Introduction In Microfluidics And Bioanalytical Applications

As newly emerging analytical technique,droplet-based microfluidics possesses many advantages in the applications of biosensing:droplet with tiny volume results in less reagent and sample consumption,as well as lower cost;high efficiency of mixing within droplet accelerates the rates of biological and chemical reactions,which shortens the time of the analysis process;the uniform size and monodispersity of droplet offers the identity of parallel experiments,which is favor of high throughput analysis;excellent sealing performance of droplet guarantees the stable reaction conditions.However,it is still facing many challenges for the application of droplet-based microfluidics in bioanalysis,such as the expensive instruments,complicated manipulations,low efficiency of analysis,complex sample introduction and cumbersome sample changing.Moreover,to realize bioanalysis in droplet-based microfluidics,it is necessary to develop suitable and compatible biosensing strategy as well.A candidate strategy not only should meet the requirements of high sensitivity and selectivity,but also take the advantages of droplet and avoid increasing the difficulties of chip design and droplet manipulation.On the basis of above considerations,to develop the droplet-based bioanalytical methods with simplicity,high efficiency and low cost,we made some efforts mainly in the following aspects.1)A novel technique of droplets manipulation was presented in droplet-based microfluidics.With this technique,the droplets were manipulated with the assembly-line mode,so we called it,"assembly-line manipulation of droplets(ALMD)".As a proof-of-concept application,the technique of ALMD was used for simultaneous multiplexed DNA detection in the microfluidic chip based on graphene oxide-assisted DNA sensing strategy.The results revealed that the simultaneous multiplexed DNA analysis was successfully carried out with the advantages of significantly time-saving and low reagent consumption.In addition,this technique of droplets manipulation is favour of improving the throughput of droplet analysis and experimental efficiency with simple chip design and instrument.2)A novel and simple droplets dosing strategy was developed based on the adsorption and desorption of polydimethylsiloxane(PDMS)surface.In daily experiment,the double-stranded DNA(dsDNA)intercalating reagent(SYBR Green I)was easily adsorbed on the microchannel wall and contaminate the channel,which affected the experimental results.Utilizing this negative phenomenon,the strategy of contact-induced droplets dosing(CIDD)was presented.For droplets dosing,SYBR Green I was firstly adsorbed on the channel wall of dosing region.When the droplets containing dsDNA passed through the dosing region and contacted with the channel wall,SYBR Green I was transferred from the channel wall into dsDNA droplets rapidly.As a result,significant fluorescence responses of droplets were observed.Since this CIDD-based microfluidic platform is capable to perform a variety of biosensing through translating into the detection of dsDNA.Therefore,the analytical targets,ranging from toxic metal ion to DNA,and to protein were successfully quantified with satisfactory results.3)A simple,flexible and low-cost sample-introduction technique was developed and integrated with droplet-based microfluidics.The sample solution was first stored in a container equipped with a homemade PDMS adaptor.Under the driving of positive pressure,the sample solution was rapidly delivered into the microchip from the container.This sample-introduction technique was so compatible that could be integrated with T-junction,flow-focus or valve-assisted droplet microchips.And the convenient sample changing was easily achieved,which laid the foundation for rapid and multiplexed detection in droplet-based microfluidics.With the proposed technique of sample-introduction,the concentration gradient generation and quantum dot(QD)-based fluorescence barcoding,as well as high-throughput droplet screening were preliminarily demonstrated.More importantly,multiplex DNA assay was successfully carried out in the microfluidic system.4)To develop sensitive and homogeneous biosensing strategy that compatible with droplet-based microfluidics,a novel composite reagent consisting of dsDNA templated copper nanoparticles(CuNPs)and SYBR Green I was prepared and exploited for the detections of H2O2 and related biomolecules.It was found that the small molecule SYBR Green I was adsorbed on the surfaces of CuNPs,instead of intercalating into the dsDNA.However,when the solution of hydrogen peroxide(H2O2)was added into the composite reagent,the CuNPs was deconstructed;meanwhile,the inhibition of SYBR Green I binding with dsDNA was eliminated,resulted in obvious change of fluorescence.Therefore,many related biomolecules could be detected through translating into the detection of H2O2.Taking advantages of the interactions of oxidases with their corresponding substrates,the detections of glucose and cholesterol were successfully carried out.Since horseradish peroxidase(HRP)could be used for catalytic decomposition of H2O2,the proposed method was also employed for HRP detection,which revealed the potential application of the method in fluorescent immunoassay.5)Based on the above efforts in the construction of biosensing strategy,a dumbbell-shaped DNA molecule was designed as multifunctional template for selective formation of fluorescent silver nanoclusters or copper nanoparticles.Benefiting from the smart design and facile synthesis,and assisted with DNA tool enzymes,a simple,label-free and homogeneous fluorescence strategy for adenosine triphosphate(ATP)detection is proposed.The strategy exhibits high sensitivity for ATP assay with a detection limit of 81 pM.Moreover,the results also illustrated the practicability and versatility of the method.To further improve the sensitivity and stability of the detection method,on the basis of the assembly of dumbbell-shaped DNA,the fluorescence emission of SYBR Green I intercalating into dsDNA instead of the fluorescence of metal nanomaterials was employed as the signal readout for ATP detection.The detection limit of the improved method was 4.8 pM.Combining the improved strategy of ATP detection with above mentioned techniques of droplets dosing and sample-introduction,the sensitive detection of ATP in droplet-based microfluidic platform was successfully carried out.