Peptide Nucleic Acid Probe-based Biosensors And Their Applications In Rapid Detection Of Disease Biomarkers

Early screening and rapid diagnosis of diseases provide us abundance pathologicalinformation to characterize malignant signatures at early stage,develop personalized treatment plan,determine therapy response and assess efficacy.A biomarker is a biological molecule found in blood,body fluids or tissues that is a sign of a normal or abnormal process,or of a malignant disease such as cancer.Typically,a series of alterations in biomarkers differentiate an affected patient from a person without the disease.There are tremendous variety of biomarkers,including nucleic acids(DNA,micro RNAs or other non-coding RNA),proteins(enzyme,receptor,antibody),polysaccharides,peptides,small molecular metabolites,cytokines,hormones,and even the whole cells(CLCs).Clinical molecular diagnostics and biomarker discoveries in pathology are advancing rapidly as we begin to understand the complex mechanisms that transform a normal cell into an abnormal one.These discoveries have fueled the development of novel drug targets and new treatment strategies since biomarkers can be easily assessed non-invasively and serially.The standard of care for patients has shifted away from an empirical treatment strategy based on the clinical-pathological profile to one where a biomarker driven treatment.Therefore,developing rapid,accurate and stabilized analytical methods possess the practical and clinical importance for real-time analysis.With biocompatibility,nanoscale control lability and highly specific Watson-Crick base pairing capability,DNA has proved to be a versatile material for programmed construction of different DNA-based biosensors.However,conventional biosensors based on molecular self-assembly,including molecular beacon,hybridization chain reaction(HCR),and walkers are limited by low specificity,complex sample treatment,poor reproducibility and high costs.Herein,peptide nucleic acid(PNA),a non-natural and uncharged analogue of DNA and RNA,has became a positive alteration for sequence-specific detection in gene expression and target analysis.It is capable of forming Watson-Crick double helices with complementary PNA,DNA and RNA.Meanwhile,special backbone properties such as uncharged,high rigidity,unnatural endow PNA with distinct chemical,physical and biological features,boosting its highly promising application both in vivo and in vitro.This thesis focuses on the development of PNA-assisted biosensors for sensitive and specific detection of biomarkers and explore the interaction between PNA and nanomaterials.The main research contents are summarized as follows:(1)While significant progress has been made in developing sensors for the detection of proteins,it is still very difficult to apply the sensors in resource-limited settings.Thus,the development of on-site and real-time colorimetric sensing platforms that are convenient,economical and sensitive is of considerable importance.Currently,numerous aptamer-based sensors are attracting increasing attention for the detection of proteins using either optical or electrochemical methods.Since aptamer is a kind of ss DNA,it can bind PNA to form a stable duplex structure following Watson-Crick hydrogen bond pairing rules.With the use of thrombin as an example analyte,we report a colorimetric aptasensor for thrombin based on the binding of Di SC₂(5)to PNA/TBA₂₉hybrids,in which Di SC₂(5)was used as a chromogenic agent and TBA₂₉was used as a thrombin ligand.The aggregation of Di SC₂(5)in the minor groove of the PNA/TBA₂₉duplex leads to a peak at 534 nm and thus the solution is purple.In the presence of thrombin,TBA₂₉binds to thrombin and releases the aggregation state of Di SC₂(5)and the solution turns blue.In this sensor,a PNA probe without any modification(AAC CTG CCC T),partially complementary to TBA₂₉,was designed and synthesized by the method of solid-phase peptide synthesis.This simple,fast,label-free,and sensitive colorimetric assay for thrombin in aqueous solution provides a promising approach for the analysis of target proteins in resource-limited settings.(2)By using a 36-bases long DNA strand with 7 pyrimidine bases at both ends and a 7-bases long homopurine PNA strand,we demonstrated the formation of a hairpin structure with a PNA-DNA₂triplex stem.Furthermore,we find that the cyanine dye Di SC₂(5)can bind with high affinity to the stem and causes a noticeable color change.Then,with the use of mi RNA-21 as an example target,we designed a colorimetric,rapid,label-free and portable detection system for mi RNAs based on PNA-DNA₂triplex and Di SC₂(5),in which Di SC₂(5)was used as a chromogenic agent and the DNA strand,which is complementary to mi RNA-21 in the loop sequence,was used as a recognition probe.This method presented high sensitivity and selectivity to mi RNA-21 and overcame the drawbacks of complicated probe modification and precise temperature control procedures.The real-time signal output and naked-eye distinguish features make it quite suitable for point-of-care analysis in resource-limited settings.(3)We reported a new label-free colorimetric assay for SNP detection by taking advantages of PNA probe and hemin-SWCNTs nanocomposite with the aid of S1nuclease to cleave target DNAs.This sensing platform relies on the different aggregation or dispersion states of hemin-SWCNTs caused by ss PNA and DNA/PNA duplexes.The genetic difference can be directly distinguished by the naked eyes since the nanocomposite can catalyze TMB into color difference.With the use of single-nucleotide mutations in tumor suppressor gene TP53 as the proof of principle analyte,we achieve rapid,simple,sensitive and convenient discrimination of SNP even in the cell lysate.The employment of water-soluble hemin-SWCNTs and label-free PNA probe endow this biosensor straightforwardness and universality for SNP detection.