| Background:In clinical medicine,blood transfusion has become an important medical method,in which safety is the top issue.Blood matching test is the essential way to guarantee the safety of blood transfusion in modern medical laboratory.In a blood matching test,an erythrocyte sample from a donor and a serum sample from a receiver are cross-mixed to check whether there is an antibody in the serum of the receiver which might destroy the erythrocyte from the donor.Only in the case when there are no detectable mismatching antigens and antibodies in the blood samples of the receiver and the donor,in another word,there are no hemolysis and precipitation,the blood from the donor can be transfused into the body of the receiver.However,problems exist in clinical blood matching test,such as,extensive labour is involved and the degree of automation remains low;on the other hand,automated blood matching instruments are expensive and costly to operate,and the consumption of reagents and samples are high,and it still relies on operators' judgment in the case of doubtful samples.As a result,the efficiency and accuracy of the blood matching equipments are not ideal.Among the recent technical developments in immune diagnosis,point-of-care diagnosis drew great attentiosn due to the low involvement of labour,ability to operate and record automatically,and moreover,miniaturization of immune diagnosis platform may benefit the realization of point of care.Among the attempts,introducing microfluidic technology,which is prone to miniaturization and automation,into the blood matching test,may benefit the realization of automation in the test and pave a way to largely decreasing the consumption of samples and reagents.Supermagnetic beads have become an important solid-state sample carrier in immune detection,such as enzyme-linked immunosorbent assay(ELISA)due to their large surface area/volume ratio and straightforwardness of being manipulated by external magnetic fields.However,the assay is lengthy and during it process,complex and repeated operations,such as reaction,blocking,separation and rinsing are required and an automation in its operation is strongly recommended.Therefore,it would be greatly meaningful to promote the efficiency of ELISAif an automated protocol with which multiple-step sample-handling procedures can be established.Objective:This study aims at establishing a microfluidic droplet-based platform for immune diagnosis and sample treatment,on which blood samples and immune reagents commonly used in conventional blood cross-matching tests,are loaded in micro-droplets.By ultilizing the automated manipulation function of digital microfluidic systems,cross-match tests of red blood cell samples and sera are carried out.As a result,the feasibility of conceptually conducting point-of-care immune diagnosis is to be conducted.Besides,supermagnetic beads which are used as solid-state carriers of immune samples are loaded in droplets.Combining with the manipulability and the delivery ability of magnetic beads,and coupling with bead anchoring module,the possibility of establishing automated multi-step sophisticated operational protocol in the sample treatment process in immune detection is to be pursued.Methods:1.A fully functional droplet-based microreactor was constructed with a suitable insulation recipe and a droplet modification method being developed for real biological and chemical samples in a blood matching test.2.The blood samples from inpatients who received blood cell transfusions in Dalian Central Hospital from April to May,2016 were chosen in the test and a record of positive antibody response prior to blood transfusion were excluded.Totally 32 blood samples were adopted.Among these,21 were from male patients,11 were from female patients and their ages were from 28-78;and the blood types were 8 in A,12 in B,10 in O and 2 in AB,and all of the blood samples were positive in Rh D.By utilizing the automated droplet manipulation functions,blood matching on microfluidic chips was conducted and the fused droplets of red blood cells and serums were observed to check whether there is any precipitation caused by interactions between antigens and antibodies.The results were compared with those obtained from the saline water method.3.A manipulation module with which magnetic beads can be anchored were built and bead-based microscale reaction,blocking,separation and rinsing were investigated.Results: 1.Digital microreactors,in which droplets were manipulated on a microfluidic chip,were constructed.Suitable chip preparation recipes and droplet modification methods were obtained and the biofouling of chips surfaces which was caused by protein-containing droplets was eradicated.2.Blood matching test on digital microfluidic chips was attempted and the results were in consistent with those from the conventional tests.And it took only 8 seconds for a positive result to show up on a microfluidic chip.The sample consumption was only 1.2 ?l,which is 1/20-1/100 of the typical sample consumption in the traditional method.3.By placing magnetic beads in droplets and being assisted by the magnetic bead anchoring module,we are able to combine the droplet manipulation platform with magnetic microbead techniques and realize the efficient separation between beads and droplets,reactions and washing of beads with buffer droplets.Based on these,we are able to automatically conduct the multiply steps of reaction,separation and rinsing on digital microfluidic chip.And these steps are the basic operations in magnetic bead-based heterogeneous ELISA.Conclusion 1.In this project,digital microfluidic droplet microreactors,in which the microfluidic chip was the key component,was established.The basic manipulation functions of digital microfluidic chips on droplets,such as transportation,fusion,separation and splitting,were achieved.2.Blood matching tests were conducted on the surface of digital microfluidic chips and the results were in consistent with those obtained from the traditional methods.And the reaction duration and sample consumptions were largely shrinked.These merits can be attributed to miniaturization of the scale of the droplet microreactors and the establishment of the droplet manipulation system.And the concept of applying digital microfluidic chips as the platform for immune precipitation reactions were approved.3.By loading the magnetic beads in droplets,the combination of the digital microfluidics system and the magnetic microbead anchoring module were made.Preliminary multiple-step reaction,separation and rinsing of bead-supported samples were realized and a protocol of carrying multiple-step sample-treatment process of an enzyme-linked immunosorbent assay in an automated was established. |
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