Construction Of Novel Test Strips And Their Applications In The Point-of-care Testing Of Small Molecule Substances

Point-of-care testing(POCT),as a simple,rapid and accurate means of obtaining detection results,is very suitable for the rapid detection of targets in resource-limited environments.In POCT,the test strip is the easiest to operate and requires the simplest equipment,which is widely used in daily life.For example,p H test strips can quickly detect p H and pregnancy test strips can help people judge early pregnancy.At the same time,test strips have also played a great role in the diagnosis of some sudden diseases.For example,under the outbreak of Covid-19,the research and development of novel coronavirus test strips and their application in the market have enabled the public to determine whether they are infected with the novel coronavirus in time,so as to take effective treatment and epidemic prevention measures.The test strips mainly include Lateral flow assay(LFA)test strips and ordinary test strips.LFA strips are widely used for qualitative and semi-quantitative analysis of biomarkers in blood,urine,saliva and other samples,but they are mostly used for POCT of macromolecular substances,while small molecules are rarely detected.The main problem is that some small molecules do not have recognition units such as antibodies and aptamer,which makes it difficult to construct conventional LFA strips.Common test strips are mostly dry strips dipped with indicators or reagents,which can be used to test the acidity and alkalinity of the solution and the presence of certain compounds,atoms and ions,such as litmus test strips,phenolphthalein test strips,p H test strips and blood sugar test strips.Common test strips have low sensitivity and can only achieve qualitative or semi-quantitative detection of small molecules.In order to solve the above problems,a novel LFA test strip with molecularly imprinted polymers(MIPs)as the recognition unit was designed to realize the POCT of thiodiglycol(TDG),which is the metabolite of mustard gas.And the triple signal amplification strategy was further used to improve the sensitivity of the peroxide strip and realize the rapid and sensitive detection of adenosine triphosphate(ATP).The detailed work is as follows:(1)A novel lateral flow assay test strip based on moleculally imprinted polymers for the point-of-care testing of thiodiglycol.We developed a novel MIPs based LFA strategy to achieve rapid detection of small molecule substance TDG.Gold nanoparticles(Au NPs),MIPs and metallothioneins(MTs)were loaded onto the conjugate pad,T ine and C line,respectively.When TDG was added,it first reacted with Au NPs via the Au-S interaction.Then,under capillary force,the TDG on the surface of Au NPs could specifically bind to the three-dimensional cavity of the MIP on the T line,and the remaining Au NPs bound to the MTs on the C line,showing two distinct red bands on the T line and the C line,respectively.In contrast,in the absence of TDG,only one red band can be observed on the C line.Thus the visual detection of TDG was realized.Furthermore,TDG could be quantitative detected through the change of T line gray value.There was an excellent linear relationship between(G_C-G_T)/G_C and the TDG concentration in the range from 10.0 pg/m L to 10.0μg/m L,and the limit of detection(LOD,3σ/k)was 0.41 pg/m L.This method has been successfully applied to the detection of TDG in human urine and is expected to be used in the rapid clinical diagnosis of sulfur mustard poisoning patients.(2)The triple signal amplification strategy based peroxide test strip for the point-of-care testing of adenosine triphosphate.We constructed a triple signal amplification strategy by using hybridization chain reaction(HCR),liposome amplification and color algorithm amplification to amplify the signal of peroxide test strip(PTS)for highly sensitive detection of ATP.Firstly,two aptamers(Apt1 and Apt2)of ATP were designed,and magnetic beads(MBs)were connected to Apt1 to form MBS-Apt1complex by the binding effect of biotin and streptavidin.In the presence of ATP,MBs-Apt1 and Apt2 can recognize ATP together to form sandwich structure.Apt2 extended a sequence that acts as an initiator to trigger the HCR of hairpin DNA H1 and H2,resulting in a long double-stranded DNA structure.In addition,one end of H1 extended a branch chain,which could hybridize with the liposome connected with single stranded DNA(ss DNA),so that multiple liposomes were connected to the long double-stranded structure of the whole DNA.Because liposomes ruptured under the action of Triton X-100 and released a large amount of the inclusion glucose oxidase(GOD),which reacted with glucose to produce a large amount of hydrogen peroxide.Then PTS could be used for detection,and the qualitative detection of ATP could be realized through the depth of blue in the strip.Finally,images were collected by smart phone and imported to computer.And the color analysis method developed by us was adopted for algorithm amplification processing.The quantitative detection of ATP was realized through the change of B value in RGB value before and after adding ATP.The experimental results showed that in the range of 50 to 250 nmol/L,there was a good linear relationship between?B and ATP concentration,and the limit of detection(LOD,3σ/k)was 0.65 nmol/L.In conclusion,in this thesis,we constructed a new MIPs based LFA test strip for the specific detection of TDG with a wide linear range,realizing LFA for small molecule substances without aptamers or antibodies.Combined with triple signal amplification strategy,the high sensitive PTS detection of ATP was realized,which solves the limited problem that ordinary test strips could only achieve qualitative or semi-quantitative detection.The above methods are universal and can be used to detect other small molecules by changing the template molecule of MIPs and replacing the DNA sequence.