Electrochemical Biosensing Technology For Detection Of Proteins And Nucleic Acid Based On Proximity-dependent Surface Hybridization Assay

With the implement of the Human Genome Project and the development of the functional genomics and proteomics research, it's an important domain that high sensitive assay methods were developed for the fast detection of the proteins and DNA in real time in the after-genome age. Electochemical biosensors attract significant attention and become the research hotspot for their simple, sensitive, selective and compatible with microfabrication technologies, et al.In this thesis, a series of novel electrochemical biosensor methods were developed to satisfy and achieve the need of the high sensitivity, selectivity and affinity, as well as prove a high performance platform for the detection of proteins and DNA. The results primarily proved that the proposed technology is reasonably comparable with the classical detection method, indicating the practicability of using the proposed method in clinical diagnosis. The detailed content described as follows:(1)In chapter 2, we exploited aptamer that was short DNA or RNA oligonucleotides selected by SELEX with high affinity for the target proteins to developed a surface proximity-dependent hybridization electrochemical aptasensor method for the high sensitive detection of model analyte PDGF-BB based on the proximity effect, that is to say a pair of affinity aptamer probes simutineously recognize the target molecules and form the proximity probes so that some complementary sequence hybridization enhanced the stability. the aptasensor was constructed by self-assembly of a short thiolated DNA oligonucleotide 1 on the gold electrode via the alkanethiol moiety at the 5'-terminal. A DNA aptamer 2 to PDGF-BB with a sequence extension at the 3'-end is used as the affinity probe. The aptamer probe has a electroactive ferrocene-labeled tail sequence that is complementary to the surface-tethered DNA strands with a predesigned low melting temperature. When aptamer pairs simultaneously bind to the homodimer of PDGF-B, the tail sequence are brought into close proximity with their local concentration increased substantially to allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. CVs, DPVs and impedances were used to systemly characteristic this method, conforming the mechanism of the biosensor. In conclusion, this method can be implemented to the high affinity detection of PDGF-BB, dynamically increased DPV current with increasing PDGF-BB concentration ranging from 1.0 pg/mL to 20 ng/mL, with a readly achieved detection limit of 1.0 pg/mL.(2)Whereas the aforementioned technology refered pair of proximity probes simultineouly to hybridizing with the surface-tethered DNA, it was found from the experiment that the proximity-dependent surface hybridization could not sufficiently improved the thermal stability and favorably enhanced the sensitivity. In Chapter 3, based on the mentioned mechanism of proximity-dependent surface hybridization assay, an electrochemical aptasensor based on the proximity-dependent surface hybridization assay by pair of different aptamer probes simultaneously bind to the protein for the detection of the related model protein-PDGF-BB was ulteriorly developed. The aptasensor interface was fabricated by self-assembly of a short thiolated DNA oligonucleotide 1 on the gold electrode via the alkanethiol moiety at the 5'-terminal. DNA aptamer 3 and 4 to PDGF-BB with a sequence extension at the 3'-end and the 5'-end is used as a pair of affinity probes. One aptamer probe has a electroactive ferrocene-labeled tail sequence at the 5'-end that is complementary to the surface-tethered DNA strands with a predesigned low melting temperature. Samely, the other aptamer probe with different recognization location has a tail sequence at the 3'-end that is complementary to the surface-tethered DNA strands with a predesigned low melting temperature. These two different aptamer probes could simultaneously bind to the different location of PDGF-BB homodimer. The tail sequence are brought into close proximity with their local concentration increased substantially to hybridized each other and allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. As a result, this method was proved to be wider detection range from 0.1 pg to 50 ng and lower detection limit of 0.1 pg.(3)In chapter 4, proximity-dependent surface hybridization assay was developed to detect DNA by electrochemical biosensor method based on the high affinity and sensitivity of proximity-dependent surface hybridization assay in order to extending the application range. Different to the proteins, the nucleic acid detection based on proximity-dependent surface hybridization exploited the target DNA per se to construct a pair of proximity probes. So an oligonucleotide as the detection probe has a electroactive ferrocene-labeled tail at the 3'-end that is complementary to the target nucleic acid with a predesigned low melting temperature by 5'-end fragment. In the presense of target DNA, the detection probe and the target nucleic acid formed the hybridization complex. The hybridization one side was the detection probe that near to the other sequences of the target and make it hybridize to the surface-tethed oligonucleotide. This draw the ferrocene closed to the electrode surface and regenerated a readily detectable signal. Compare with the conventional sandwich hybridization assay, this approach ensured the ferrocene marker to enough close the electrode surface and increased the charge transport efficiency as well as improved the sensitivity remarkably. The detectable range dynamictally was from 1 fM to 1 nM and in the range the peak current exhibits a linear correlation to the log of target DNA concentration, the limit of the detection is 1 pM. This electrochemical DNA sensor can be regenerated and discriminate the mismatch with different bases sequences.(4)In chapter 5, because of the aptamer unsatisfied the need of plentiful proteins detection, electrochemical immunosensor based-on proximity-dependent surface hybridization assay was used to detect the proteins. Herein a pair of antibody- modified oligonucleotids replaced the aptamer probes extend proximity-dependent surface hybridization assay to the proteins immunoassay that might creat a universal methodology for developing high-performance biosensors in sensitive detection of proteins. Electrochemical immunosensor based on proximity-dependent surface hybridization assay for high sensitive PSA detection was proposed. Similarly to the electrochemical aptasensor mechanism in chapter 3, a pair of mono-antibodies recognized different antigenic determinants of PSA were used to covalently crosslink two oligonucleotides. One probe has a electroactive ferrocene-labeled tail sequence at the 3'-end that is complementary to the surface-tethered DNA strands at 5'-end with a predesigned low melting temperature. Samely, the other probe has a tail sequence at the 3'-end that is complementary to the surface-tethered DNA strands at 3'-end with a predesigned low melting temperature. Furthermore, the first probe at the backward tail sequence and the seconde probe at the foreward tail sequence have a short complementary region with a low melting temperature. Antibodies simultaneously bind to the antigene, two oligonucleotides tail sequences are brought into close proximity with their local concentration increased substantially to hybridized each other and allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. The PSA was determined in the range of 25 pg to 1 ng with the detection limit of 25 pg and the immunosensor could be reusable.(5) In chapter 6, a new sensitive, selective, reagentless and reusable electrochemical DNA sensor for detection of hybridization based on electrochemical channels switched by allosteric molecular beacon was described. This electrochemical DNA sensor employs hairpin oligonucleotide labeled with biotin as a block at one tail and labeled with NH2 at the other tail that interacted with disulfide heterocyclic compound to fix on the gold electrode surface. Because molecular beacon-like DNA stem duplex has the biggest space hindrance and the biotin as a block interacted with the 11-mercaptoundecanoic acid (MUA) increase the space hindrance, the electroactive molecules could not commendably diffuse on the electrode surface. When the probes hybridized with the target nucleic acid, the block was moved away from the electrode surface and thus formed an electrochemical pinhole channel in the 11-mercaptoundecanoic acid (MUA) self-assemble film. So the electroactive molecule can diffused, regenerated the electrochemical redox signal. The DNA target, associating with theα-thalassemia gene containing the codon 142, was determined in the range of 2.8×10-18~8.7×10-8 M, and the detection limit can be reached 1.1×10-19 M. This electrochemical sensor can be realized the high sensitive, selective, reagentless and reusable DNA biosensor and uses for indentication of SNPs.(6) In chapter 7, a novel electrochemical immunosensor was established to detect h IgG based on gold nanoparticles tag and enhancement by surface absorbtion voltaggram assay. h IgG as the model analyte, the immunosensor interface was fabricated by immobilized GAH IgG on the glass carbon electrode surface. The immobilized antibody reacted with the analyte target-the h IgG and form sandwich configuration by using the colloidal gold-label goat anti-human immunoglobulin G (GAH IgG) as the detection probe. The size of gold nanoparticles and the apparent area of the electrode was enhanced by nanogold catalyting the reduction of HAuCl4 on the electrode surface. The self-asseble absorption of electroactive probes on the gold nanoparticles surface was exploited to measure the absorption amount on the electrode surface by DPV. The developed method is expected to hold great promise in immunosensing due to the ease of implementation, high sensitivity and specificity.