Smartphone-imaged And Battery-powered Electrophoresis Titration Chip For Alkaline Phosphatase Assay In Serum By Moving Reaction Boundary

As a vital enzyme in organisms,alkaline phosphatase（ALP）catalyzes the dephosphorylation of various substrates,such as nucleic acids,proteins,alkaloids,etc.,showing great clinical significance in diagnoses of bone or liver cancer,bone metastases,rickets,and extrahepatic biliary obstruction.In recent years,numerous methods have been advanced for the assay of ALP in blood samples,tissues and cells,including colorimetry,electro-chemiluminescence,chemiluminescence,and fluorescence.Those methods have satisfactory stability,sensitivity,automation and high throughput.However,most of them depend on the specialized instruments or detectors,such as spectrophotometer,electrochemical transducer and fluorophotometer,which is bulky,expensive and inaccessible.There is still no really portable chip for ALP assay in blood.Herein,the main purposes of this work are as follows:（1）Novel theory and device for ALP catalysis-electrophoresis titrationA cellphone-imaged and battery-powered electrophoresis titration（ET）model for really portable assay of ALP activity based on moving reaction boundary（MRB）.In the cathode,ALP catalyzes the dephosphorylation of4-MUP yielding the product of 4-MU with one negative charge（[4-MU]^-）,which emits strong blue fluorescence under UV excitation.After applying the electric field,[4-MU]^-migrates from the cathode to the anode due to its negative charge,meets the hydrogen ion and forms a moving reaction boundary（MRB）.The velocity of MRB(V_MRB)has a positive correlation with ALP activity.Thus,ALP activity of the unknown samples could be determined through the standard curve between V_MRB and ALP activity.The whole ALP-ET system is quite simple,portable and low-cost,consisting of a UV flashlight,a home-made PMMA microchip,a pair of platinum electrodes and a lithium cell.（2）Validation of ALP catalysis-electrophoresis titration methodTo demonstrate the validity of ALP-ET method,a series of systematic experiments were conducted.The results showed that the catalysis product of ALP entered the microchannel under the electric field,reacted with hydrogen ion and created a MRB denoted by visible blue fluorescence.And V_MRB was linear to ALP activity with a linear range of 1.0-10.0 U/L:V_MRB=0.033A+3.87,R²=0.9980,showing a good linearity.The built method had a satisfactory sensitivity（LOD is 0.1 U/L）,selectivity,precision（RSDs=2.7⁶.8%）and a good recovery of 101¹05%.In addition,the stability of the lithium cell was also examined.Results showed that when the cell was continuously worked for more than 12 hours,the obtained electric field could remain more than 98%of initial voltage,indicating less than 2%decrease of cell voltage.With 180 channels for the simultaneous runs of ET assay,the electric field was still stable（more than 98%initial voltage）for60 minutes.Within 2 min run,there were 99.67%initial voltage for the ET run with 180 channels.The titration run could be completed within 2minutes,meaning that the lithium cell was quite stable in ALP-ET method.It was calculated that one could achieved a stable ET for more than 100hours with aid of voltage stabilizing circuit for the cell.（3）Application of ALP catalysis-electrophoresis titration methodTo evaluate the analytical reliability and clinical application,the proposed method was employed for ALP activity detection in human serum.Based on the standard curve between V_MRB and ALP activity,ten human serum samples were detected by the ALP-ET chip.At the same time,the same samples were measured via the classic method of microplate reader.As shown by comparative results,the analytical values of ALP activities by ALP-ET chip were close to those by the classic method.Statistical analysis implied no significant difference between the ALP activities measured via the ET chip and those detected via the classic method.Also,It was notable that the ALP-ET chip was capable of distinguishing normal adults from the patients with liver diseases.As a common inhibitor of ALP,sodium orthovanavate was chosen as for inhibitor assay investigation on account of its antineoplastic activity.Results revealed that MRB velocity declined with the increasing of inhibitor amount,showing a negative linear correlation.The regression equation was V_MRB=-0.016C_in+1.942（R2=0.9933）with a detection limit of 0.1μM,indicating a good sensitivity.As demonstrated by these results,the developed ALP-ET also offered an opportunity for further investigation of ALP inhibitory effect in anti-cancer drug discovery as well as visual and portable orthovanavate assay.