Electrogenerated Chemiluminescence Biosensor For The Determination Of Metal Ions

Lead ion is a toxic metal ion due to its causing a number of adverse health effects.Once introduced into the body,lead ion is a potential neurotoxin that can cause chronic inflammation of the kidney and heart,inhibit brain development,and decrease nerve conduction velocity.Mercury ion,the most stable form of inorganic mercury,is highly toxic environmental pollutant.And microbial biomethylation of Hg2+ yields methyl mercury that accumulates in the body through the food chain,and is known to cause brain damage and other chronic diseases.It is highly desirable to develop sensitive methods for the detection of Pb2+ and Hg2+.DNA was shown in 1994 to carry out catalytic functions and thus became the newest member of the enzyme family after proteins and RNA.Since then,the DNA molecules(called DNAzymes)have been shown to catalyze many of the same reactions as RNA or protein enzymes.When compared with RNA and protein enzymes,DNAzymes are relatively less expensive to produce and more stable to hydrolysis.Unlike proteins,most DNAzymes can be denatured and renatured many times without losing binding ability or activity.Most DNAzymes require metal ions for their activities,and some even show metal specificity.Therefore,DNAzymes can be used for metal sensing.The covalent attachment of metal complexes to oligonucleotides,which formed "metal-base pair",has been reported in various contexts such as energy transfer or electron transfer through DNA,construction of synthetic endonucleases,and as end-bound nodes for the assembly of artificial DNA constructs.Among them,the thymine-Hg2+-thymine(T-Hg2+-T)coordination chemistry and the resulting Hg2+-stabilized hybridization of oligonucleotides with T-T mismatches,have been demonstrated as a selective and versatile strategy for Hg2+ detection.ECL biosensors combine the advantages of both electrochemical and chemiluminescent biosensors,such as high sensitivity,ease of control and using of simple equipment and specific selectivity offered by the biological recognition elements,which has widely been used in pharmaceutical analysis,bioanalysis,environmental analysis and clinical analysis.The aim of this thesis is to design and fabricate ECL biosensors for determination of metal ions and lectin with high sensitivity,selectivity and regeneration.In this thesis,taking advantages of the unique properties of dendrimer and the specificity of biological molecular recognition elements,such as DNAzyme,T-Hg2+-T construction and mannose/Con A,we have designed two types of"signal on" and label-free ECL biosensors for determination of Pb2+.Two types of multi-label and multi-load ECL biosensors for determination of Hg2+ were fabricated.In addition,a ECL biosensor based on target-induced displacement for determination of Con A using mannose as molecular recognition element was fabricated.The thesis includes two parts.First part,chapter 1,is general introduction while second part consisting of five chapters,is a research report.In Chapter 1,general introduction to biosensor and research development of metal ions sensing methods and strategies for advancement of ECL biosensors,and the purpose of this research work were presented.In Chapter 2,a highly reproducible and sensitive signal-on ECL biosensor based on the DNAzyme for the determination of lead ion was developed.The ECL biosensor was fabricated by covalently coupling 5'-amino-DNAzyme-tagged with ruthenium bis(2,2'-bipyridine)(2,2'-bipyridine-4,4'-dicarboxylic acid)-ethylenediamine(Rul-17E')onto the surface of graphite electrode modified with 4-aminobenzoic acid,and then a DNA substrate with a ribonucleotide adenosine hybridized with Rul-17E' on the electrode.Upon binding of Pb2+ to the Rul-17E' to form a complex which catalyzes the cleavage of the DNA substrate,the double-stranded DNA is dissociated and thus led to a high ECL signal.The signal linearly increases with the concentration of Pb2+ in the range from 5.0 to 80 pmol/L with a detection limit of 1.4 pmol/L and a relative standard derivation of 2.3%.This work demonstrates that using DNAzyme tagged with ruthenium complex as an ECL probe and covalently coupling method for the fabrication of the ECL biosensor with high sensitivity,good stability and significant regeneration ability is promising approach.In Chapter 3,a novel label-free ECL biosensor for the determination of lead ion is designed by employing DNAzyme as molecular recognition element and Ru(phen)32+ as an ECL signal compound.The biosensor was fabricated by coupling 5'-ethynyl-DNAzyme(5E')to a 4-azidoaniline(4-AA)using a copper(I)-catalyzed "click reaction",following the 4-AA was attached to the surface of the glass carbon electrode(GCE)by electrochemical reduction of the diazonium cation generated in situ from the 4-AA,and then hybridizing a DNA substrate(5DS)with a ribonucleotide adenosine(rA)with 5E' on the electrode to form a double-stranded DNA(5E'·DS).In the absence of Pb2+,Ru(phen)32+ intercalated into the grooves of 5E'·DS,led to produce a high ECL signal.In the presence of Pb2+,upon binding of Pb2+ to the 5E'·DS to form a complex(5E'·DS·Pb2+),this complex catalyzed the hydrolytic cleavage of the 5DS at the site of rA and the 5E'·DS was dissociated to form a flexible single stranded DNA,allowing Ru(phen)32+ to be released from the 5E'·DS on the surface of GCE modified with 4-AA,and thus led to a low ECL signal.The result showed that the ECL signal linearly decreased with increasing the concentration of Pb2+ in the range from 20 pmol/L to1.0 nM with a detection limit of 7.2 pmol/L and a relative standard derivation of 2.9%.The ECL biosensor was highly reproducible and possessed long-time storage stability and fast response.This work infers that using Ru(phen)32+ intercalation and "click reaction" for the fabrication of the ECL biosensor with high sensitivity and significant regeneration ability is promising approach for the design of label-free ECL biosensor for the determination of other metal ion.In Chapter 4,a multi-label ECL biosensor for highly sensitive detection of mercury ion was developed on basis of mercury-specific oligonucleotide(MSO)served as molecular recognition element and bis(2,2'-bipyridine)-4'-methy-4-carboxybipyridine ruthenium complex(Rul)as an ECL signal complex.The pendant(dend-Rul)was prepared by covalently coupling Rul with the generation fourth polyamidoamine(G4-PAMAM).The ECL probe((CH)6-oligo(ethylene oxide)-MSO-dend-Ru 1)was prepared by covalently coupling 3' phosphate group(CH2)6-oligo(ethylene oxide)-MSO with the dend-Rul.The ECL biosensor was fabricated by covalently coupling the ECL probe onto the surface of glass carbon electrode modified with single-wall carbon nanotubes through a spacer of 5'-amino-(CH2)6-oligo(ethylene oxide).Upon binding of mercury ion to thymine(T)bases of the MSO on the(CH)6-oligo(ethylene oxide)-MSO-dend-Rul,the T-Hg-T structure was formed and thus the MSO changed from linear configuration to "hairpin" configuration and the ruthenium complex was closed to the surface of the electrode,resulted in a high ECL intensity.The newly developed biosensor showed a high reproducibility and possessed long-term storage stability(92.3%initial ECL recovery over 30 day's storage).The ECL biosensor showed an extremely low detection limit of 2.4 pmol/L Hg2+.This detection limit was about 2?3 orders of magnitude lower than that reported on the basis of electrochemical biosensors and fluorescent and colorimetric methods.The effect of length of the spacer and the surface density on the ECL response was investigated.A binding constant of 8.05±0.3×108gmol-1L between Hg2+ and(CH2)6-oligo(ethylene oxide)-MSO-dend-Rul was estimated using an ECL based Langmuir isotherm approach.This work demonstrates that using G4-PAMAM as a carrier of ruthenium complex and molecular recognition element for the fabrication of ECL biosensor with sensitivity is a promising approach.In Chapter 5,a multi-load ECL biosensor for highly sensitive detection of mercury ion was developed on basis of mercury-specific oligonucleotide(MSO)served as molecular recognition element and bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine ruthenium complex(Rul)as an ECL signal complex.The ECL biosensor was fabricated by covalently coupling the ECL probe onto the surface of glass carbon electrode modified with G4-PAMAM and single-wall carbon nanotubes.Upon binding of mercury ion to thymine(T)bases of the MSO,the T-Hg-T structure was formed and thus the MSO changed from linear configuration to "hairpin" configuration and the ruthenium complex was closed to the surface of the electrode,resulted in a high ECL intensity.The ECL biosensor showed an extremely low detection limit of 0.04nmol/L Hg2+.A binding constant of 3.82±0.3×108mol-1L between Hg2+ and MSO-Rul was estimated using an ECL based Langmuir isotherm approach.This work demonstrates that using G4-PAMAM as modification electrode materials for the fabrication of ECL biosensor with sensitivity is a promising approach.In Chapter 6,a novel ECL biosensor for sensitive detection of Concanavalin A(Con A)using mannose as a molecular recognition element and ruthenium bis(2,2'-bipyridine)(2,2'-bipyridine-4,4'-dicarboxylic acid)-ethylenediamine(Rul)as an ECL signal complex was develop.The ECL biosensor was fabricated by covalently coupling mannose onto the surface of gold electrode modified with p-phenylenediamine(p-PD)using glutaraldehyde as a cross-linker and a,?-diamine-polyethylene glycol(PEG)as a spacer.And then the Con A tagged with Ru1 bind the sites on the mannose immobilized on the electrode.The introduction of target Con A induced the displacement of Con A tagged with Rul from the mannose on the electrode and thus led to a low ECL signal.The result showed that the ECL signal linearly decreased with increasing the concentration of target Con A in the range from 6.6 x 10-10 mol/L to 6.6 x 10-7 mol/L with a detection limit of 2.7 x 10-10 mol/L and a relative standard derivation of 2.5%.This work also demonstrates that the target-induced mannose displacement strategy and covalently coupling method for the fabrication of the ECL biosensor is promising approach in the design of an ECL biosensor for the determination of other lectins.In conclusion,this work demonstrates that using G4-PAMAM as a carrier of ruthenium complex and modification electrode materials and using "click reaction" and covalently coupling method for the fabrication of the ECL biosensor with high sensitivity,good stability and significant regeneration ability are promising approach.In addition,this work also demonstrates that using the target-induced mannose displacement strategy and covalently coupling method for the fabrication of the ECL biosensor in the interaction between carbohydrate and lectins provide a platform.