Designing And Researching The Nano-biosensor Based On Electrochemiluminescence

Nucleic acid,protein and enzyme are the most important biomolecules in life. Nucleic acid is in charge of transferring genetic information and coding other biomolecules,while protein has run through the whole life process.Enzyme plays an important role in DNA synthesis and maintaining the integrity of genome.The replication,repair and recombination of nucleic acids are essential life processes. DNA/RNA structure fragment,as "bricks" of the biomacromolecules,is an important participant in the activities of life and is an important signal transduction of cells.The completion of the related function of gene needs the cooperation of the protein, enzyme and the small bioactivity molecules.Actually,the matching and the effect between these biomolecules construct the basic of the vital phenomena,such as growth, generation and metabolism.The study of functional gene,protein and the related bioactivity molecules is the hot spots of postgenome era.With the increasing knowledge about human diseases,the specific sequence of DNA,correlative protein,enzyme and small bioactivity molecule detection received increasing attention.Many detection techniques of DNA sequence and protein have been developed in recent years,but there is still a lot of dissatisfaction.The radioactive labels present many problems such as a potential hazard to analyst and environment. Non-radioactive labels such as fluorescent,chemiluminescent and biotin-avidin label probes also present many shortcomings such as low sensitivity or complex equipment or others.So it is necessary to develop another method for the more sensitive, easy-to-use,fast,inexpensive detection of correlative bioactivity molecules to adapt to wide-scale genetic and protein testing requires.Electrochemiluminescence(ECL),the generation of an optical signal triggered by an electrochemical reaction,has attracted much attention during the past several decades due to its versatility,simplified optical setup,very low background signal, good temporal and spatial control,and has become an important and valuable technique for immunosensing,DNA hybridization assays and proteinic biosensors.It also provides a powerful research tools and methods for the life sciences entering into the molecular level field.Today,nano-science and technology has entered the limelight and has been investigated extensively by governments and scientists all over the world.It is out of question that the revolution of nanotechnology is coming.In recent work,it has been discovered that materials in the nano-size scale(1～100 nm)display size-dependent optical,magnetic,electronic and chemical properties.Except for these,nanomaterials have unknown chemical and physical properties that differ greatly from the bulk substances.Therefore,nanoparticles can be applied to many fields,such as optical devices,electronic devices,catalysis,sensor technology,and biomolecular labeling, etc.,which is also the reason that a burst of research activities have been focused on nanoparticles.Modified electrodes based on nanomaterials combined with high surface area and good electrocatalytic abilities that can largely improve electrical responses and the detection sensitivity.At the same time,the bioactivity of the biomolecules can be remained preferably.Today,many wonderful nanostructured materials have been applied in electroanalytical chemistry and some important progresses along these topics have recently achieved.The goal of the present study is to design and research novel DNA,protein and enzyme nano-biosensor with high sensitivity and selectivity.This paper combines the excellent characteristics of nanoparticles,the specific recognition of molecular recognition elements and the electrochemiluminescence technique.It would broaden the research field of post-genetic time.This paper altogether divided into five parts. The details are given as follows:Part one:Preface(Chapter 1)In this chapter,we elaborated review from the principles,characteristics of the electrochemiluminescence,the two most primary types of ECL reaction and their application in analytical chemistry field.The principles,characteristics and the research progress of DNA and the aptamer electrochemiluminescent biosensor all had been introducded.Nanoparticles,especially the application of the variety of nanomaterials in the electrochemiluminescent biosensor were presented.Finally, expounded the aim and the significance,pointed out the research content and the innovation in this paper.Part two:Detection of thrombin using electrochemiluminescence based on Ru(bpy)₃²⁺-doped silica nanoparticle aptasensor(Chapter 2,3)Tri(2,2'-bipyridyl)ruthenium(â…¡)-doped silica nanoparticles(Ru(bpy)₃²⁺-SiO₂ NPs) were prepared by water-in-oil(W/O)microemulsion method.A great deal of Ru(bpy)₃²⁺was immobilized inside the nanoparticle,which could greatly enhance the ECL response and result in the increased sensitivity.In chapter 2,sensitive and selective aptasensor using Ru(bpy)₃²⁺-SiO₂ NPs as DNA tags for detection of thrombin was developed based on the target protein-induced strand displacement of the DNA probe.For the proposed aptasensor,the aptamer was assembled on the surface of the Au electrode through Au-S binding.The hybridization event between the DNA probe labeled by the Ru(bpy)₃²⁺-SiO₂ NPs and the aptamer was evaluated by ECL measurements.Then,the DNA probe was displaced by thrombin and the binding event between the thrombin and the aptamer was monitored by ECL measurements again.The difference of ECL intensity(ΔI_ECL)of the two events could be used to quantify the thrombin.Other proteins,such as bovine serum albumin and bovine hemoglobin,had almost negligibleΔI_ECL.Under the optimal conditions,theΔI_ECLwas linearly related to the concentration of the thrombin in the range of 10 fM to 10 pM and the detection limit was down to 1.0 fM since SNPs containing a large number of Ru(bpy)₃²⁺molecules were labeled on the DNA probe.In chapter 3,a new electrochemiluminescent detection system for protein using the aptamers was developed.Two different aptamers,which recognize different positions of thrombin,were chosen to construct sandwich type sensing system for protein,and one was immobilized onto the gold electrode for capturing thrombin onto the electrode and the other was used for detection:To obtain the signal,the aptamer for detection was labeled with Ru(bpy)₃²⁺-SiO₂ NPs.The increase of the ECL signal generated by Ru(bpy)₃²⁺-SiO₂ NPs was observed in dependent manner on the concentration of thrombin added.Bovine serum albumin and bovine hemoglobin had almost negligible responses.The ECL signal was linearly related to the concentration of the thrombin anatyte in the range of 2.0 fM to 2.0 pM and the assay allowed detection at levels as low as 1.0 fM of the thrombin.Part three:A controllable solid-state Ru(bpy)₃²⁺-electrochemiluminescence film for detection of biomolecules based on conformation change of ferrocene-labeled molecular beacon(Chapter 4,5 and 6)In chapter 4,a controllable solid-state electrochemiluminescence film based on efficient and stable quenching of ECL of Ru(bpy)₃²⁺by oxidizing ferrocene(Fc)at the electrode was developed.The ECL intensity was correlated to the distance which was controlled by the conformation of the ferrocene-labeled DNA molecular beacon (Fc-MB)between the Fc and Ru(bpy)₃²⁺immobilized on the electrode.The conformation adjustment was conducted via complementary DNA's hybridizing with the bases in the loop of the Fc-MB and changing the temperature of the Fc-MB and the resultant double strand DNA(dsDNA).Those events all resulted in change of the ECL intensity.Therefore,the melting temperature of the relevant biomolecules was predicted successfully.With such characteristics,the solid-state Ru(bpy)₃²⁺-ECL film had the potential to calculate thermodynamic parameters of equilibrium constants of MB binding and the stem-loop formation.Such controllable solid-state Ru(bpy)₃²⁺-ECL film could detect target ssDNA,and the analysis results were sensitive and specific.In chapter 5,we utilized the special solid-state Ru(bpy)₃²⁺-ECL film to develop solid-state electrochemiluminescence biosensing switch.Thrombin as one important physiological protease in blood had been chosen as the target for investigating the application of the biosensing switch system to special protein.Herein,the loop bases of the Fc-MB were designed with the specific anti-thrombin aptamer sequence,which was expected to transform into G-quartet structure to combine with thrombin. Consequently,the Fc was far away the electrode surface and the ECL intensity was increased significantly.The enhanced ECL signal was expected to quantify the thrombin.Other proteins,such as bovine serum albumin and bovine hemoglobin,had almost negligibleΔI_ECL.TheΔI_ECLwas linearly related to the concentration of the thrombin analyte in the range of 10 fM to 10 pM and the assay allowed detection at levels as low as 1.0 fM of the thrombin.The biosensing switch system could apply to the mixed protein samples.In chapter 6,we further utilized the special solid-state Ru(bpy)₃²⁺-ECL film to develop solid-state electrochemiluminescence biosensing switch.T4 DNA ligase was chosen as the target herein for exploiting the switch system's application for biosensing ligase.The ligation system was composed of a DNA ligase,two oligos to be ligated and a MB,in which the combined sequences of the two oligos were complementary to the loop sequence of the MB.In the beginning,each oligo was hybridized to one-half of the loop of the MB to form a DNA complex with a nick.This would not open the MB stem completely,but would slightly destabilize the stem.When ligase was added, the ligation reaction closed the nick to form a longer DNA strand that was complementary to the MB,resulting in the MB opening completely and the Fc being pulled away from the electrode.Thus,the difference of ECL intensity before and after the ligation(ΔI_ECL)could use to quantify the T4 DNA ligase with sensitivity and selectivity and the detection limit was down to 2.5 U/L.Part four:Ultrasensitive detection of adenosine using solid-state electrochemiluminescent sensing platform based on structure-switching signaling aptamer(Chapter 7)In chapter 7,in the present study,a solid-state electrochemiluminescent sensing platform based on structures-witching signaling aptamers for highly sensitive detection of small molecules wss developed using adenosine as a model analyte.A gold electrode was first modified with cysteamine and Ru(bpy)₃²⁺-AuNPs composite.Then, ferrocene-labeled aptamer was assembled onto the modified electrode surface via Au-S interaction.The hybridization event between the ferrocene-labeled aptamer and the complementary ssDNA sequence was evaluated by ECL measurements.Then,the introduction of adenosine triggered structure switching of the aptamer.As a result,the complementary ssDNA sequence forced to dissociate from the sensing interface, resulting in a decrease in ECL intensity.The decrement of ECL signal(ΔI_ECL)of the two events was proportional to the amount of adenosine.The present sensing system could provide both a wide linear dynamic range(10 nM to 10μM)and a low detection limit(5.0 nM).In addition,high selectivity,good reproducibility,stability,and reusability were achieved.The recovery test demonstrated the feasibility of the designed sensing system for an adenosine assay.Part five:Enzyme-amplified electrochemiluminescence detection of p53 sequences using MWNTs-Ru(bpy)₃²⁺aggregates(Chapter 8)In chapter 8,an effective solid-state electrochemiluminescent biosensor had been developed for the detection of human tumor suppression p53 gene.Positively charged Ru(bpy)₃²⁺could be immobilized stably on the electrode surface with negatively charged carboxyl of multi-wall carbon nanotubes(COOH-MWNTs)in the form of aggregate via electrostatic interaction.MWNTs-Ru(bpy)₃²⁺composite and ssDNA labeled by the AuNPs were linked together through pyrrole polymer membrane(PPy). AuNPs were favorable candidates for the immobilization of enzymes because amine groups and cysteine residues in the enzymes were known to bind strongly with AuNPs. Moreover,MWNTs and PPy could act as tiny conduction centers to facilitate the transfer of electrons.Such biosensor combined enzymatic selectivity with the sensitivity of ECL detection,and it displayed wide linear range,high sensitivity and a low detection limit(0.1 pM).The mutant type p53 DNA sequence could be obviously distinguished from the wide type p53 DNA sequence.