| The early diagnosis of malignant tumors and other major diseases has been an important research focus.If a major disease could be screened out and thus ascertained at its early stage,it can be subjected to a timely and effective treatment so as to greatly improve the survival of patients and reduce the medical cost as well.So,the development of sensitive,selective,operationally simple,and cost-effective methods for the detection of their biomarkers is of great importance to the diagnosis and prognosis of human diseases.Electrochemical DNA biosensors are such tools,through which the target single-stranded(ss)or double-stranded(ds)DNA fragments can be specifically recognized and electrochemically quantified using a sequence-known capture probe according to the Watson-Crick base-paring rule.They have attracted considerable attentions in many areas,such as biochemical analysis and precision medicine,by virtue of their low detection cost,operational simplicity,high selectivity and sensitivity,good portability,high amenability to miniaturization,etc.Although great progress has been made in many aspects,current electrochemical DNA biosensors are still suffered from the drawbacks such as poor stability and specificity of the applied capture probes,limited detection sensitivity,complicated preparation process,and lengthy turnaround time,from the viewpoint of clinical applications.Taking advantage of peptide nucleic acid(PNA)as the capture probe,this dissertation focuses on surmounting the abovementioned drawbacks,which is expected to can greatly improve the practicability of electrochemical DNA biosensors.The dissertation includes 1)the fast and highly selective electrochemical detection of ssDNA based on the carbodiimide chemistry-mediated conjugation of organometallic compounds;the highly sensitive and selective electrochemical detection of ssDNA using the 2)polysaccharide-or 3)biomimetic catalysis-mediated in situ metallization as a signal amplification strategy;4)the highly sensitive and selective electrochemical detection of ssDNA using the surface-initiated electrochemically mediated atom transfer radical polymerization(SI-eATRP)as a signal amplification strategy;and 5)the highly sensitive and selective electrochemical detection of dsDNA using the strand replacement of dsDNA by PNA and the SI-eATRP as a signal amplification strategy.1.Electrochemical detection of ssDNA based on carbodiimide chemistry-mediated conjugation of organometallic compoundsFirstly,thiol-terminated PNA(HS-PNA)probes were immobilized onto gold electrode through gold-sulfur selfassembly for the specific recognition of target ssDNA(tDNA)fragments.After hybridization,NH2-containing OMCs,aminoferrocene(AFc),were directly conjugated to the free 5,-terminal phosphate groups,mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)and imidazole,of the captured tDNA fragments.As the captured tDNA fragments are labeled with AFc in a stoichiometric ratio of 1:1,the measurement of the proportionally conjugated Fc probes based on differential pulse voltammetry(DPV)enables a quantitative analysis of tDNA.Mediated by the carbodiimide(CDI)chemistry,OMCs can be covalently conjugated to the 5'-terminals of the captured tDNA fragments in a one-step manner and served as the electroactive probes,offering the benefits of easy operation,low cost,and high efficiency.Under optimal conditions,this method allowed a fast detection of tDNA within the linear range from 0.1 nM to 100 nM(R2=0.998),with a detection limit of 93 pM(S/N=3).A"signal-on"method like this is highly immune to false-positive results.Moreover,it shows great potential toward genotyping of single nucleotide polymorphisms(SNPs),and holds high anti-interference capability in the presence of complex serum samples.Therefore,combined with the use of PNA as the specific capture probe,the method based on the direct conjugation of electroactive OMCs via the CDIs chemistry to the terminals of the captured target ssDNA fragments can be used to the fast and highly selective electrochemical detection of ssDNA.2.Electrochemical detection of ssDNA based on polysaccharide-mediated in situ metallizationFirstly,HS-PNA probes were immobilized onto gold electrode through gold-sulfur selfassembly.After hybridization,polygalacturonic acid(PGUA)molecules were attached to the PNA/DNA heteroduplexes via the phosphate-Zr4+-carboxylate chemistry.Next,the vicinal hydroxy groups of the polysaccharide backbones were cleaved and oxidized into aldehyde groups by sodium periodate(NaIO4).The resulting aldehyde groups acted as reductants and converted added silver ions into silver particles,which in situ deposited onto the electrode surface.Finally,the deposited silver particles were electrochemically stripped off and measured by DPV for the quantitative analysis of tDNA.Compared with those approaches involving the use of natural enzymes or nanoparticles for in situ metallization,the polysaccharide-mediated in situ metallization-based signal amplification strategy is low-cost and operationally simple.Moreover,as a kind of renewable biomaterials,the use of polysaccharides as signal amplification mediators offers the benefits of broad availability and environmental friendliness.Under optimal conditions,this method allowed a quantitative analysis of tDNA within a linear range from 0.1 fM to 10 pM(R2=0.998),with a detection limit as low as 2.5aM(S/N=3).Again,this is a"signal-on"method,which is highly immune to the false-positive results.Moreover,it shows great potential toward genotyping of SNPs,and holds high anti-interference capability in the presence of complex serum samples.Therefore,the combined use of PNA as the specific capture probe and the polysaccharide-mediated in situ metallization as a signal amplification strategy is applicable to the highly sensitive and selective electrochemical detection of ssDNA.3.Electrochemical detection of ssDNA based on biomimetic catalysis-mediated in situ metallizationFirstly,HS-PNA probes were immobilized onto gold electrode through gold-sulfur selfassembly.After hybridization with tDNA fragments,hematin molecules were attached to the hybridized PNA/DNA heteroduplexes via the phosphate-Zr4+-carboxylate chemistry.Then,the attached hematin molecules acted as biomimetic catalyst in accelerating the reduction of silver ions in the presence of catechol,leading to the in situ deposition of silver particles onto the electrode surface.Finally,the deposited silver particles were stripped off and measured by square wave voltammetry(SWV),through which the amount of tDNA can therefore be quantitatively analyzed.Compared with the direct use of natural enzymes for signal amplification,the biomimetic catalysis-based strategy can be featured by the advantages of low cost and high stability.Under optimal conditions,there was a good linearity between the stripping current and the logarithm of tDNA concentration over the range from 0.1 fM to 0.1 nM(R2?0.998),with a low detection limit of 62.41 aM(S/N=3).Results also demonstrate that this method is applicable to the genotyping of SNPs and holds high anti-interference capability in the presence of complex serum samples as well.Therefore,the combined use of PNA as the specific capture probe and the biomimetic catalysis-mediated in situ metallization as a signal amplification strategy is applicable to the highly sensitive and selective electrochemical detection of ssDNA.4.Electrochemical detection of ssDNA based on surface-initiated electrochemically mediated atom transfer radical polymerizationFirstly,HS-PNA probes were self-assembled onto gold electrode for the capture of tDNA fragments.After hybridization,ATRP initiators,a-bromophenylacetic acid(BPAA),were attached to the hybridized PNA/DNA heteroduplexes via the phosphate-Zr4+-carboxylate chemistry,followed by the growth of electroactive polymers,with ferrocenylmethyl methacrylate(FMMA)as the monomer,via the surface-initiated electrochemically mediated atom transfer radical polymerization(SI-eATRP)at a negative potential.The formation of polymers leads to the labeling of numerous electroactive probes,which in turn greatly improves the resulting electrochemical signal.Compared with the direct use of preformed polymeric materials for signal amplification,the electrochemically mediated in situ polymerization-based strategy can greatly improve the coupling rate and coupling efficiency of polymeric chains on electrode surface.Under optimal conditions,it allowed a quantitative analysis of tDNA within a broad linear range from 0.1 fM to 0.1 nM(R2=0.996),with the detection limit down to 0.072 fM(S/N = 3).Compared with the CDIs chemistry-based method,more than 1.2 × 106-fold improvement in detection sensitivity can be achieved.Results also indicate that it shows great potential toward the genotyping of SNPs,and holds high anti-interference capability in the presence of complex serum samples.Therefore,the combined use of PNA as the specific capture probe and the SI-eATRP as a signal amplification strategy is applicable to the highly sensitive and selective electrochemical detection of ssDNA.By virtue of its operational simplicity,high efficiency,and cost-effectiveness,the SI-eATRP-based signal amplification strategy holds great potential in bioanalytical applications for the sensitive detection of biological molecules.Moreover,the high compatibility of the in situ growth of polymers by potential control with microfabrication technology makes it an ideal solution for the high-throughput detection on microelectrode arrays.5.Electrochemical detection of dsDNA based on strand replacement of dsDNA by PNA and SI-eATRPSimilarly,HS-PNA probes were self-assembled onto gold electrode surface for the specific recognition of target dsDNA fragments.The PNA probes firstly bind to the ends of their complementary strands in the dsDNA fragments through end invasion,followed by the migration of PNA probes along the complementary strands and the complete replacement of the other strands by PNA probes,leading to the capture of their complementary strands onto the electrode surface.Thus,the strand replacement of dsDNA by PNA can bring a high density of phosphate groups on the electrode surface for the attachment of ATRP initiators,BPAA,Via the phosphate-Zr4+-carboxylate chemistry.The growth of electroactive polymeric chains,with FMMA as the monomer,via the SI-eATRP was achieved at a negative potential.Compared with the triplex-forming oligonucleotides(TFOs)-based methods,which are essentially restricted to the use of homopyrimidine/homopurine oligonucleotides,this method is applicable to the direct detection of mixed-base dsDNA.In addition,the formation of polymers leads to the modification of numerous electroactive probes,thereby greatly improving the electrochemical signal.Under optimal conditions,a good linearity between the electrochemical signal and the logarithm of target dsDNA concentration over the range from 1.O fM t0 1.0 nM(R2=0.997),with a detection limit of 0.47 fM(S/N=3),can be obtained.A"signal-on"method like this is believed to be highly immune to false-positive results.Results demonstrate that it has high selectivity for dsDNA detection,and holds high anti-interference capability in the presence of complex human serum samples.Therefore,this method can be applied to the direct,highly sensitive,and highly selective electrochemical detection of dsDNA. |
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