Development Of A New Micro Free-Flow Electrophoresis Apparatus And Study Of Its Application In Detection Of Glucose

Free-flow electrophoresis （FFE） is electrophoresis technology used forcontinuous separation of biology sample, which is based on aqueousenvironment and can be used for analysis and preparation. As backgroundbuffer and samples enter into separation chamber, the samples will beseparated according to their electrophoretic mobility under the effect ofelectric field which is perpendicular to flow direction. FFE has been widelyused in biology field for its merits of continuous separating, mild reactioncondition and high recovery. After years of development, FFE has formedbranchs of free-flow zone electrophoresis （FFZE）, free-flow isotachophoresis（FFITP）, free-flow isoelectric focusing （FFIEF） and the free-flow field step electrophoresis （FFFSE）. The FFZE mode is widely used.The micro free-flow electrophoresis （μFFE） has been given moreattentions since Raymond present an μFFE device firstly in1994. Unlikelarge FFE device, μFFE device can conduct a separation in very short time,and only requires a microliter amount of the sample. The μFFE technologygets high attention in the past20years in the field of separation science.However, the μFFE apparatuses in current are mainly made by PDMS,silicon or glass which are not only expensive, but also complicated tomanufacture. As the apparatus can not be reused, and others disadvantageslike low voltage efficiency and sealed electrode chamber, the bubbles can notbe discharged timely, resulting in a bad sample stream in separation cavity.To this end, we have developed a novel μFFE device, and the size ofseparation chamber is45mm×17mm×100μm （volume of77μL）. Thevolume of the separation chamber is much smaller than ordinary FFEapparatus. It has6inlets and12outlets, and a "sandwich-like" structure isformed by a ion permeable membrane between upper and lower PMMAplates. Both electrode chambers are open-design and can effectively removebubbles produced by electrode, so as to reduce the impact of bubble onsample stream in separation chamber. The experiments show that:1) due tothe use of ion-permeable membrane with high conductance, the voltage utilization is significantly improved to90.5%;2) the apparatus can be reused,which can effectively solve the clogging problems;3) open-electrodechamber can effectively reduce the impact of electrolysis bubble onseparation chamber.As reported before, μFFE device is usually used for separation, ratherthan online detection of biological samples, let alone the actual sample. Tomeet the requirements of online testing for biological samples in laboratory,we construct a supramolecular interaction system in μFFE device. Theinteraction of boronic acid-functionalized benzyl viologen （o-BBV, acts likequencher） and8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt （HPTS,acts like fluorescent dye） will result in the quenching of HPTS. The glucosecan react with the complex of o-BBV/HPTS and the fluorescence of HPTSwill be restored。In the experiment, the glucose will be added in backgroundbuffer and the sample is the complex of o-BBV/HPTS. Images will becaptured with a fluorescence microscopy and further analyzed withMetaMorph software. As the FFE device is transparent, optical on-devicedetection of fluorescent complex during the electrophoretic separation isfeasible. The experimental data show that there is a linear regression equationfor the change in fluorescence intensity and glucose concentration (F/F0=1.697+0.0386×C_glucose，R²=0.95) in the glucose concentration range of1×10^-8 M to5×10^-7M. And this device is also used for the online detection of theglucose in actual urine sample, which further prove the usability of thedevice.