The Application Of Distance And Temperature Based Signal Readout Methodology For POCT

Traditional detection methods are generally based on the combination of relevant theories on optics,electromagnetism or electrochemistry with specific principles of analytical chemistry to meet the detection requirements for target samples.These methods have the advantages of precise detection and good versatility,whereas,they are often expensive with the requirement for professional operators and high maintenance cost,which are not favorable to portable detection and limit their popularization.At the same time,with the rapid development of modern science and technology and the improvement of human life quality,people are increasingly concerned about their life and health and propose a higher demand for the rapid and accurate medical diagnosis,thus creating point-of-care testing.The so-called point-of-care testing(POCT)is a new detection method that field test can be realized quickly at the site of a disease or an event without complex processing and operation.POCT with the advantages of simple operation,low consumption,low cost,portability and so on,is a newly-developing field of laboratory medicine.As a new type of means of medical diagnosis,POCT fully satisfies the requirements of high efficiency and fast pace in today's society,and has become more and more widely used in clinical diagnosis,food security and public security,etc.Aptamer and microfluidics have been.widely used in POCT after rapid development in past thirty years.Aptamer with the advantages of high specificity,strong binding affinity,good stability and no immunogenicity,as well as microfluidics with the advantages of miniaturization,integration and automation,highly satisfy the intrinsic requirement of POCT.Making full use of the advantages of both avails the further development of POCT.Enzyme-linked immunosorbent assay(ELISA),is a classical detection method based on the immobilization of soluble antigen or antibody on the solid phase carrier and the specific immunoreaction between antigen and antibody for quantitative or qualitative detection.As a commonly used detection method,ELISA with the advantages of high sensitivity,strong specificity,good commonality and so on,is often used in POCT.However,it still suffers from the tedious repeating sampling and washing steps in practice.Therefore,the integration of ELISA operation is of great practical significance and also can better serve the POCT.Analysis above,the work of this thesis mainly has two aspects:1.Visual and portable detection of distance based signal readout.Visual detection is the simplest signal output mode in POCT,which meets the requirements of POCT well.With the help of specific target recognition and flexible signal transduction of DNA Hydrogel,highly selective and visual detection can be realized.DNA Hydrogel is a kind of colloidal solid formed by the cross-linking among the two polyacrylamide chains modified with DNA-A and DNA-B sequence respectively and Aptamer-Linker strand that can partly hybridize with A and B sequences.Upon introduction of a target to bind with Aptamer-Linker specifically,it will compete with DNA hybridization,and then the gel will collapse,resulting in the release of signal molecules or signaling molecules for signal amplification and readout.Herein,cocaine is selected as the model target and Platinum nanoparticles(PtNPs)are pretrapped in the hydrogel.In the presence of cocaine,it will bind with Linker,which causes the gel to collapse and release the trapped PtNPs.Then the gel supernate was added to the simply built detection device,so that the PtNPs can catalyze hydrogen peroxide to produce oxygen that could push the dye moving inside the device.Finally,the concentration of target molecule is calculated by measuring the dye moving distance by a ruler.Using the device,a linear detection range was obtained from 0 to 200 ?M of cocaine in the PBS buffer,and the detection limit of cocaine is 5.6,uM in 50%urine.As a portable test method,further detection of adenosine also demonstrates its versatility.2.Portable detection of integrated ELISA of temperature based signal readout.As a common precious metal,gold is extremely chemically stable.Gold nanorod is a kind of gold nanoparticle ranging from several nanometers to hundreds of nanometers dimensionally.Gold nanorods(AuNRs)not only inherit the chemical properties of its bulk materials,but also show good optical and photothermal properties.Compared with spherical gold nanoparticles,AuNRs have stronger surface plasmon resonance(SPR).Upon near infrared laser radiation,the strong absorption properties of AuNRs makes light energy be converted into thermal energy efficiently,and the longitudinal SPR absorption peak of AuNRs can be regulated by controlling the aspect ratio.Based on these excellent properties,AuNRs have many potential applications in optical imaging,photothermal therapy and photothermal sensing,etc.In this thesis,seed-induced growth method was adopted to synthesize AuNRs with high aspect ratio,then the AuNRs were modified with biotin in order to introduce them into ELISA reactions easily.And then,with the aid of magnetic manipulation of magnetic beads,the integration of the whole ELISA reactions in PMMA(polymethyl methacrylate)chip was realized by controlling the magnet outside the chip.At last,the reaction liquid was exposured under the laser irradiation of 850 nm,and the concentration of target protein was calculated by measuring the temperature change of the reaction liquid using a digital thermometer.Based on the model target of c-reactive protein(CRP),the integration of tedious operation of traditional ELISA and the purpuses of fast and cheap test and simple operation had been realized in this thesis.