Development Of The Novel Paper-based Analytical Devices And Their Applications In Point-of-care Testing

Point-of-care testing（POCT）,referring to a detection way that is not limited by time and place,is carried out at the sampling site and uses portable analytical devices and corresponding reagents to quickly obtain the detection results.Due to the characteristics of miniaturization of analytical equipment,simplification of operation assays and timely report of results,it has become one of the most important research fields of analytical sensing research in recent years.Paper-based analytical devices show their unique advantages in POCT because of their low cost,easy modification and good portability,and are widely used in the fields of environmental monitoring,food safety and clinical diagnosis.However,due to the low sensitivity and accuracy caused by external environmental factors and complex matrix effects,the use barriers of ordinary users,the need for external equipment for result reading and so on,which restrict the wide application of paper-based analytical devices in POCT.Focusing on the development and application of the novel paper-based analytical device,this paper puts forward a series of effective strategies to improve its POCT performance.The specific research contents are as follows:1.A novel paper-based analytical device by enzyme-mediated sodium alginate hydrogel is proposed for point-of-care detection of organophosphorus（Ops）pesticides with distance readout.With the aid of acetylcholinesterase-mediated sodium alginate hydrogel,this method converts the solution viscosity to the diffusion distance on the paper.The inhibition effect of acetylcholinesterase by organophosphorus pesticides causes the p H of the system to decrease and the amount of Ca²⁺released decreases,following to that the viscosity of the alginate hydrogel on paper decreases and the diffusion diameter increases.The concentration of Ops is quantitatively determined by ruler-measurable diffusion diameter on the paper without detection equipment.Our proposed assay exhibits excellent reproducibility and good selectivity for detection of Ops with the limit-of-detection（LOD）of 3.3 ng/m L.This study provides a new strategy for POCT application and the development of paper-based analytical device based on distance readout by using enzyme inhibitor to adjust solution viscosity.2.On the basis of the first work,considering that a single detection signal on paper-based sensor is easy to be disturbed by the external environment,which reduces the sensitivity and accuracy of the method,we constructed a dual-readout paper-based sensor for point-of-care detection of penicillinase.With the aid of penicillinase-mediated alginate hydrogelation,there is changes both the viscosity and p H of the reaction solution.Drop the solution on a mixed cellulose ester filter paper for sensing,which is coated with a color indicator.Dual signals of the targets are easily achieved from the sensing spot on the paper since the modulations of viscosity and p H of the reaction solution would result in changes of diffusion diameter and color of the spot,respectively.A smartphone-based readout device is developed to record and analyze both the diffusion diameter and color signal for quantitative detection of penicillinase.The wide linear range(8.00×10^-3 to 2.67 m U/μL for distance-readout and 8.00×10^-2 to 2.67 m U/μL for color-readout)with low limit-of-detection(2.67×10^-3 m U/μL for distance-readout and 2.67×10^-2 m U/μL for color-readout)have been achieved using the dual-readout paper-based sensors.The proposed method is successfully applied for the detection of penicillinase in milk samples,showing its potential value for analysis of complex real samples.3.Another important factor that reduces the accuracy and sensitivity of paper-based analysis device is the influence of complex matrix effect.Here,we propose a colorimetric paper-based analytical device（c-PAD）based on alginate hydrogel for point-of-care detection of whole blood samples.With the aid of alginate hydrogel with a nano-scale network structure,enzyme（such as GOx,HRP）and chromogenic reagent（such as TMB）were encapsulated into hydrogel for the color reaction of the target（such as turning the colorless TMB into a blue TMBox）.At the same time,the nanoscale size sieving effect could effectively prevent the red blood cells（7-8μm）and hemoglobin（5-6 nm）,so as to realize the in-situ non-separation detection of small biological molecules in whole blood.In addition,simultaneous detection of different targets in whole blood can be achieved by fabricating c-PAD with multiple sensing regions.Owing to its several essential advantages including extremely simple procedure for fabrication,sample-to-answer analysis without tedious pretreatment,and high-throughput analysis,the proposed c-PAD will be of great value in POCT applications of whole blood samples.This method can be extended to the detection of other small molecules in whole blood and has wide applicability.4.Biochemical analysis of whole blood samples means invasive sampling.Based on the third work,a novel non-invasive eye-patch wearable biosensor to simultaneously assay multiple biomarkers in human tears is developed.The sensor is uniquely designed as a convenient-to-use eye-patch which can be easily and comfortably fit under eyes.Different sensing regions on the eye-patch are modified with specific chromogenic reagents to selectively determine hydrogen ion（p H）,protein,ascorbic acid and glucose in tears.The assay takes only one drop of tear（～20μL）and 30-second response time.The resulting color signals from each sensing region on the eye-patch are captured and analyzed by a smartphone,and a cloud model based on the Internet of Things（Io T）concept is recommended which can extend the application of the eye-patch biosensors in point of care testing and health monitoring,especially during the pandemic.Tear detection of the human subjects is performed using the proposed method and the results display good correlation to parallel assay kits measurements.The results show the great potential and versatility of the proposed eye-patch biosensor for non-invasive determination of diverse tear biomarkers,facilitating the widespread application of the tear-based non-invasive wearable biosensor in emerging personal health-care and clinical monitoring fields.