Aptamer-Based Chemiluminescence Assay

DNA aptamers are synthetic single-strand nucleic acids discriminated by screening in vitro,which are of high affinity and specificity to many given targets ranging from small molecules to large proteins and even cells.Some new methods for efficient and fast detection in biochemical and biomedical fields are being developed based on the molecular recognition of aptamers.Aptamers have been demonstrated to have advantages over antibodies with regard to chemical stability,readily availability, simple modifiability,and high flexibility in biosensor design for analyte detection.As a novel functional molecular,aptamers have gained considerable interest in bio-analytical application in many fields such as clinical diagnosis,environmental supervision,pharmacal study,etc.Chemiluminescence(CL)has been exploited within a wide range of applications in various fields,due to their extremely high sensitivity along with their extra advantages such as needless special light,simple instrument,wide calibration ranges, and suitability for automatical operation in analytical chemistry.Nowadays,based on cross-research with other disciplines,CL has been successfully served for different applications.Our study developed a series of novel detection technologies on the strength of the high specifically binding between aptamers and its corresponding ligands in the thesis.And simultaneous detection of two molecules in one sample has been made grounded on aptamer switch.Description of research in the thesis is presented as follows:Chapter 1:IntroductionThis chapter is composed of two phases.The first phase addresses the source,traits, and advantages of aptamers and outlines aptamer-based detection techniques,then presents single analysis of current studies based on aptamer in detail,including optical aptasensors,electrochemical aptasensors,and other mass sensitive aptasensors. Following that,the multiplex analysis has been also stated in the development of aptamer-based biosensors and bioassay methods.The second phase of this chapter reviews current simultaneous multi-analytes detection by substrate-resolved CL technology exampled HRP and ALP in assay field.With that,objectives,significance, contents of this CL research based on aptamer are summarized. Chapter 2:Label-free aptamer-based chemiluminescence detection of adenosineOwing to no elucidation for the binding property of aptamer,it has difficult to label on aptamer and the label process would affect the binding affinity between the targets and their aptamers to a greater or lesser degree.Consequently,some label-free detection methods have been focused to develop,especially in some fileds like bioassay and environmental supervision.In this chapter,employing magnetic bead as a special biomolecule separating carrier,we demonstrate a sensitive CL aptasensor for label-free adenosine assay based on 3,4,5-trimethoxyl-phenylglyoxal(TMPG)as the signal molecule,which reacts specially and instantaneously with guanine nucleobases of adenosine-binding aptamer strands.We designed two different experimental routes for assay and the sketchy experiment steps were described as follows.(1)The magnetic beads were activated and the capture DNA immobilized on magnetic beads by the force of NH₂-COOH covalent bind.(2)Route A:The excess and quantitative adenosine aptamers reacted with different amounts of adenosine and formed an adenosine-aptamer complex.Then the rest free aptamer,along with the complex,were hybridized with capture DNA immobiled on magnetic beads.Route B:the aptamers first hybridized with capture DNA-magnetic bead conjugates and then reacted with adenosine.When adenosine as a specific competitor appeared,parts of aptamer were compelled to take apart from the magnetic beads and formed an adenosine-aptamer complex.(3)After speration by magnetic force,The CL signal was gotten from the raction between TMPG and guanine nucleobases of aptamer strands on magnetic beads.Using this system,adenosine could be specifically detected and a relatively low detection limit(8×10^-8M)as well as a wide linear dynamic range(4×10^-7-1×10^-5 M)could be achieved with route A(R²=0.9852).With route B,adenosine can be detected in a shorter range from 4×10^-7M to 5×10^-5M(R²=0.9764).Overall,the label-free protocol described here may have value in a variety of clinical,pharmacal and environmental applications for which the simple,fast and accurate quantitative analysis of biomolecular adenosine is desired.Chapter 3:Label-free aptamer-based chemiluminescence detection of PDGF-BBPlatelet-derived growth factor(PDGF)is a ubiquitous mitogen and chemotactic polypeptide present in serum for stimulating cells of mesenchymal origin and elicits multifunctional actions with a variety of cells.PDGF-BB,one of the important isoforms of PDGF,has been directly implicated in the cell transformation process and in tumor growth and progression.And the detection of PDGF-BB is of very valuable in biological fields.In this chapter,a"sandwich-type"detection strategy was employed in our design to make PDGF-BB detection.The procedure was as follows. Anti-PDGF-BB antibody immobilized on the surface of carboxyl terminated magnetic beads and DNA aptamers,both of which flanked PDGF-BB.After washing,the CL signal was obtained via the instantaneous derivatization reaction between TMPG and guanines nucleotides of aptamer to detect target protein without any label procedure. On this ground,an amplified CL approach was investigated employing G-rich sequence linked with aptamer as the amplification unit.The streptavidin was employed as an amplification platform to develop a high effective CL approach. Nonamplified approach achieved a good linear correlation from 1×10^-8to 2×10^-7M with the lowest detection concentration(1×10^-8M)and correlation coefficient (R²=0.9901).In amplified approach,an excellent linearity was found within the range of 4×l0^-10-2×10^-8M(R²=0.9954)with the lowest detection concentration of 4×10¹⁰, which was 25-fold improvement on nonamplified methods.The result demonstrated that this assay was reproducible,speedy,easy to use and suitable to make further development.In a word,this novel lable-free CL approach provided great promise for PDGF-BB assay in clinical field.Chapter 4:ATP CL determination coupled the enrichment of magnetic beads with the specific binding of aptamerThe enrichment of immuno-magnetic beads is one of simple,speedy and high efficient screening methods.For one complicate sample with trace quantities of targets,it has unique merits following as:First,The irregular shape of the magnetic beads allows for much greater surface area.The large surface area results in high binding capacities,allowing much more targets captured with increasing capture probes on magnetic beads.Second,magnetic beads with high quality have great homogenicity and paramagnetism,which guarantee smoothly reaction with biomolecular without any intervention from any particle impurity.Third,Most of the rinsing solution was removed by pipet,but some buffer liquid(³Î¼L)remained around the magnetic beads because of the force of superficial adsorption.It was almost impossible to dry the sample completely and this benefited the prolonged active of biomolecular.Greater magnetic responsiveness results in faster magnetic separations especially on high throughput automated platforms.In this chapter,we employed adenosine triphosphate(ATP)aptamer as capture probe to covalently bind on magnetic beads.ATP present in trace quantities were enriched from the mixed sample on the magnetic beads and could be rapidly isolated from the sample by employing an external magnetic field in order to make CL detection.Experimental results confirm that it has good sensitivity with a detection limit of 1×10^-8M for ATP. ATP could be specifically isolated and detected and a wide linear dynamic range (1×10^-8-1×10^-5M)could be achieved(R²=0.9918).On the whole,by using magnetic beads with great enrichment and aptamer with high discrimination,this method may show good perceptive on the purification and detection of biomolecular.Chapter 5:Simultaneous detection founded on sptasensors by CL technologyA novel aptamer-based CL biosensing platform for the simultaneous detection of two small molecules as exemplified by adenosine and cocaine has been developed. Herein,we employed magnetic bead as the special biomolecule immobilizing carrier and made CL detection catalyzed by two labeled enzymes of alkaline phosphatase (ALP)and horseradish peroxidase(HRP).The principles of homogeneous substrate-resolved technology for CL detection have three steps:(1)preparation of adenosine and cocain aptamer-linked biosensors based on magnetic beads as carriers; (2)binding with a mixture of adenosine and cocaine to initiate target-aptamer reactions;(3)reacting with a mixed solution of Digoxin-ALP and Strepavidin-HRP followed by magnetic force separation of the magnetic beads conjugates and parallel detection of CL signals from HRP and ALP with their corresponding assay kits. Experimental results confirm that this CL immunosensing platform has good sensitivity with the lowest detection concentration of 1×10^-8M for adenosine and cocaine.On the whole,by using different aptamers,this method may offer a new direction in designing high-performance CL aptasensors for sensitive simultaneous determination of small molecules.And the technique would play an important role in multiplex analysis for biomoleculars with simple instrument and convenient procedure.