Construction And Application Of Paper-based Microfluidic In Accurate Blood Gouping

Rapid and reliable blood grouping plays an essential role in various clinical medicine,forensic medicine,and genetics scenarios.Although classical tube and gel card method have the advantages of accuracy and reliability,these techniques have failed to meet the requirements of rapid and portable blood grouping in a field-deployable manner for outdoor screening and emergency rescue,which is largely due to the limitation of time-consuming and requiring complicated apparatus.In addition,weak agglutination caused by drugs,diseases and other disturbances may lead to false negative results,which brings another challenge for rapid and accurate typing.It is crucial to develop a point-of-care blood grouping strategy in complex environments.Therefore,this study constructed a new quick response(QR)code paper-based microfluidic,which proposed a triple quality control strategy for blood group analysis to solve the problem of weak agglutination blood group recognition,and realize the rapid and accurate recognition of ABO,Rh and rare blood groups under emergency conditions.The specific research is as follows:1.Construction of QR code paper-based microfluidic analysis platformWe successfully constructed the QR code paper-based microfluidic analysis platform(5×5 cm),which proposed the QR code design concept without coding by using the recognition layer,reaction layer and sample layer assembly.The chromatography calibration patterns set separately in the QR code are used as external quality control,so as to achieve direct smartphone analysis without additional accessories.The positioning area of the QR code can complete automatic positioning and angle correction.Cotton linter paper is preferred as the substrate for microfluidic because of its relatively loose and porous fiber structure.It was found that the red blood cells(RBCs)containing specific surface antigens could be captured completely by cotton linter paper,and irregular agglutinates were formed in the fiber.Compared with the commonly used 3MM chromatographic paper,the agglutinated RBCs chromatographic distance on cotton linter paper was significantly shorter(11.50±1.08mm,P<0.001),and the color intensity was significantly different(197.26±1.14)from that of unagglutinated RBCs(106.61±1.67)(P<0.001).Laser cutting was used to achieve precise paper-based microfluidic structure machining.The QR code paper-based microfluidic is composed of a recognition layer,reaction layer and sample loading layer,which is cut by laser.ABO and Rh(D)blood grouping can be realized by observing whether the color of the window turns red in the visual within 30 s.The detection accuracy of 30 clinical samples was 100%.It is proved that the QR code paper-based microfluidic has both miniaturization and high response performance,and it can be used for rapid blood grouping under emergency conditions.2.Study on the mechanism of weak agglutination blood group recognition and automatic typing strategyA blood group identification strategy based on the specific coloration of bromocresol green(BCG)dye was constructed,and the internal quality control scheme of the dye specific coloration was investigated.The results showed that BCG reacted with human serum albumin(HSA)can form characteristic teal complex,and existed characteristic peak at 630 nm.This strategy can effectively improve the specificity of blood type detection and accurately identify red samples that are similar to blood(P<0.001).A dual quality control scheme for blood group identification was proposed,including negative control and BCG induced-color change,to eliminate potential interference such as hematocrit(HCT)and paper substrate quality.Therefore,the QR code of the ABD card was successfully constructed for ABO and Rh(D)blood grouping.Developing smart phone blood grouping(BG)recognition program,which calibrates traditional red/green/blue(RGB)model by using hue/saturation/value(HSV)model,and integrate external quality control to improve the accuracy of detection.We proposed the recognition theory of weakly agglutinated RBCs based on differentiated HCT and integrated dual quality control scheme.Further,we construct an automatic blood type typing model based on machine learning(AUC=1),which can realize accurate,rapid and automatic detection of weak agglutination blood group within 30 s.And the accuracy rate was 100%after 200 clinical samples detection.The blood group results can be automatically uploaded to the database in real time,which can provide data support for pre-transfusion preparation and an optional solution for outdoor emergency testing.3.Construction of rare blood groups synchronous recognition and accurate analysis platformThe accurate classification strategy of complex environment and blood group was proposed,and analyzed the influence of plasma albumin,total bilirubin,and total cholesterol on color response.The albumin concentration was positively proportional to the color intensity,and the total bilirubin and total cholesterol had no obvious color change.And the accuracy rate of smartphone detection was 100%,under the detection conditions of different light intensity(50 Lx～5000 Lx),card rotation angle(0°～315°),and shooting angle(45°～135°).The detection process is not limited by light,angle and position,which significantly improves the detection stability of complex blood samples in complex environments.Based on this,a QR code paper-based microfluidic was successfully constructed to achieve rapid identification of multiple rare blood groups.With optimized conditions,two hundred blood samples with ABO(A/B)and Rh(D/C/c/E/e)blood groups were detected within 30 s,and fifty blood samples with rare blood groups including Rh(D/C/E/c/e),MNS(M/N/S/s),P(P1),Kell(K),Kidd(Jk~a/Jk~b),and Lewis(Le~a/Le~b)were automatically typed within 3 min.And the accuracy rate was 100%(AUC=1)compared with the gold standard.The paper-based rare blood group microfluidic proposed in this study has effectively promoted the popularization of blood group testing in resource-scarce areas,and holds great value in improving blood type information database and preventing blood transfusion reactions.The QR code paper-based microfluidic platform proposed in our study can not only detect weak agglutination blood groups,but also can classify a variety of complex rare blood groups.Stable and reliable results were achieved by integrating the triple quality control strategy,which can effectively reduce the interference of complex environment.This platform provides an alternative for outdoor emergency detection and provides data support for pre-transfusion preparation.After further development,the platform can be used as a universal platform,which is expected to be popularized in food safety,early diagnosis of diseases,environmental monitoring and other fields.