| Simultaneous detection of multiplex has gained considerable interest in clinical, environmental,and biodefense applications,etc.Numerous multianalyte assays have been developed due to its high sample throughput,short assay time,low sample consumption and reduced overall cost,as compared to parallel single-analyte assays. In recent years,considerable effort is mainly focused on developing multi-label-based multianalyte assay methods.Most of the current developed approaches for multianalyte are based on spatially resolution or multiple-labels mode.However,there are some restrictions in the number of labels and,therefore,of analytes that could be determined simultaneously.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 simple instrumentation,wide calibration ranges,and suitability for miniaturization in analytical chemistry.In contrast to current multi-label-based detection techniques,we took good advantage of three different kinds of carriers to separate targets by thermo-triggered precipitation,and one label rather than multiple was employed.According to their character in common,all of them could be seperated by centrifugation at 35℃.Meanwhile,each of them has its unique attributes;namely,they can be seperated from each by magnetic force,centrifugation under room temperature and 35℃,respectively.Description of research in my thesis is presented as follows:Chapter 1:This thesis first addresses multi-label-based detection techniques,and then presents current state of knowledge,including fluorescent-labe,enzyme-labe,etc. The second phase of this chapter reviews current main spatially resolution mode and its applications in various fields,and discusses the development in this field. Following that,objectives and significance of this research are summarized.Chapter 2:Instantaneous Derivatization Technology for Simultaneous and Homogeneous Determination of Multiple DNA TargetsThere are potential advantages,in terms of simplicity and speed,for detecting DNA hybridization steps directly without using any external labels,especially for the multiplexed assays.In the current paper,we describe the use of a carrier-resolved label-free multiplexed assay for the simultaneous detection of multiple DNA targets. Herein we demonstrate that this protocol,using three homogeneous carriers' thermosensitive poly(N-isopropylacrylamide),polystyrene beads,and magnetic beads, respectively,for simultaneous determination of three short DNA fragments specific to hepatitis B virus.Briefly,one hybridization occurs between a mixture of three different capture probe DNAs immobilized onto three carriers and three targets in a single vessel,and then chemiluminescence(CL) detection proceeds via an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxylphenylglyoxal(TMPG) and the guanine nucleotide-rich regions within the target DNA.An excellent linearity is found within the range between 0.1 and 6.0 pmol with the lowest detection limit of 100 fmol.In contrast to current encoding strategies,every hybridization signal for the corresponding DNA target in our protocol is uniquely immobilized onto one carrier vehicle with a unique and intrinsic physical-chemical signature.Moreover,an instantaneous derivatization reaction is employed for the label-free determination of three targets in a single vessel. In addition,a simple CL setup is employed to read the carrier code instead of an expensive and complicated flow cytometer or imaging system commonly used for multiplexed assays.Further signal amplification is achieved by employing three amplified DNAs for second hybridization,which include a guanine nucleobase-rich sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with one of the target DNA as an amplification platform. Such simple amplified CL transduction allows detection of DNA targets down to the 15-fmol level.This new protocol also provided a good capability in discriminating perfectly complementary DNA from single-base mismatches and noncomplementary sequences.Overall,the protocol described here may have value in a variety of clinical, environmental,and biodefense applications for which the accurate quantitative analysis of multiple DNA targets is desired.Chapter 3:Carrier-resolved Technology for Homogeneous and Simultaneous Detection of Multiple PCR AmpliconsA general purpose label-free chemiluminescence(CL) platform is developed wherein polymerase chain reaction(PCR) amplicons labeled with one hapten are detected via an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxyl-phenylglyoxal(TMPG) and the guanine nucleotides within the PCR target DNA.As a proof of principle,the response selectivity of this platform is evauated for simultaneous determination of three PCR amplicons specific to Hepatitis B Virus(HBV) by using three homogeneous carriers magnetic beads,polystyrene beads,and thermo-sensitive poly-N-isopropylacrylamide(PNIP),respectively. Briefly,PCR amplicons are labeled with digoxin,biotin or FITC via the modified up-stream primers.After PCR amplification,the immunoreactions occur between a mixture of three target PCR amplicons and three modified carriers with either anti-digoxin,streptavidin or anti-FITC in a single vessel and then each carrier is separated from the others under different conditions based on their attributes for direct CL detection.Different from conventional endpoint PCR detection techniques where dsDNA PCR amplicons have to be denatured(e.g.,heated to 95℃for a few minutes) before the detection hybridization,firstly this new protocol offers the enhanced sensitivity due to the absence of competitive hybridization.Secondly, multiplex real-time PCR required the use of multiple fluorophores with emission peaks that can be unequivocally separated,whereas herein an instantaneous derivatization reaction is employed for the label-free determination of three targets in a single vessel,and every signal for the corresponding PCR amplicon in our protocol is uniquely immobilized onto one carrier with a unique and intrinsic physical-chemical signature.Thirdly,specialized expensive equipment is required for the detection of real-time PCR amplicons whereas herein a simple CL setup was employed to perform our novel multiplexed PCR assays.Overall,the assay is sentive,safe,and cost-effetive when compared to conventional agarose gel electrophoresis,real-time PCR for the detection of PCR amplicons.Furthermore, the protocol described here may have value in a variety of clinical,environmental, and biodefense applications for which the accurate quantitative analysis of multiple PCR amplicons is desired.Chapter 4:Sequential Determination of Four Proteins by Temperature-Triggered Homogeneous Chemiluminescent ImmunoassayA novel protocol for performing a sequential multiplex immunoassay,based on a temperature-triggered separation/mixing process and alkaline phosphatase(AP) and horseradish peroxidase(HRP) catalyzed chemiluminescence(CL) detection,is described.Herein we introduce poly(N-isopropylacrylamide)(PNIP) and magnetic beads as bimolecular immobilizing carriers to separate different antigens by taking advantage of thermal response.PNIP is known to aggregate and precipitate out of water when the temperature is raised above the lower critical solution temperature (LCST) of 31℃;thus,it can be separated from supernatant by centrifugation.Besides, magnetic beads can be separated from PNIP by magnetic force as the temperature is lower than LCST.A homogeneous noncompetitive ELISA was employed,formed by using IgG,IgM,IgA,and growth hormone(GH) as two couples of carrier analytes, two couples of capture antibodies were immobilized onto the surface of magnetic beads and PNIP,respectively and AP and HRP labeled second antibodies.With a sandwich format,highly sensitive CL detection of HRP was applied,and the detection limits ofIgG and IgA were as low as 2.0 and 1.5 ng/mL,respectively.
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