Development Of Novel Electrochemical Biosensing Technology Based On Nano-materials

Electrochemical biosensors have valuable applications in bio-medicine, environmental monitor, food industry, and drug because of their high sensitivity, simple instrument, rapid response, and low cost. Use of biosensing techniques to detect the meaningful bio-markers for the accurate and rapid diagnosis of clinical diseases and heavy metals in our environment has been a novel, attractive and hot topic in the current medical studies and environmental science. But the conventional electrochemical biosensors have many problems such as low sensitivity, slow response and poor stability. In the past few years, the appearance of nanomaterials has provided a new approach for solving the above problems. Applying nanomaterials for the fabrication of biosensors would greatly improve the performance of the resulting biosensor and has become the research hotspot for their unique structure and properties. In addition, small-molecule-linked DNA has emerged as a versatile tool for the interaction assay between small organic molecules and their protein receptors. In the view of the enormous amplification detection strategies of oligonucleotides, this method is expected to hold considerable potential for the selection of clinical drugs and the proteomic researches. In this dissertation, a series of novel electrochemical biosensors strategies were developled for the detection of protein, DNA fragment and heavy metal ions with low cost, ease in operation and high sensitivity. The detailed materials are described as follows:(1) Carbon nanotubes (CNTs) have valuable applications in electrochemical biosensors due to large aspect ratio, excellent electrical conductivity and high electrocatalytic activity. In order to make CNTs very promising in the development of immunosensors for protein detection, we developed a novel electrochemical immunoassay strategy based on PL-coated MWNTs as the electrochemical labels in Chapter 2. These electrochemical labels could be easily prepared via the noncovalent modification of the MWNTs followed by covalent conjugation with antibodies. Also, these labels could be readily incorporated into a magnetic separation-based sandwich immunoassay protocol. And then magnetic separation allowed highly effective and selective accumulation of the electrochemical labels and the assembly of MWNT labels onto the C18H37SH self-assembled monolayer (SAM) mediated the redox of numerous indicator molecules with a strong redox current triggered due to the tunneling effect of the CNTs. The experiment results demonstrated that this strategy could afford superb sensitivity and exquisite specificity for prostate specific antigen (PSA) associated with tumors. The strategy was demonstrated to exhibit dynamic responses to the protein target over a four-decade concentration range from 5 pg/mL to 100 ng/mL with the detection limit of 3 pg/mL and further determine the PSA in serum samples. In view of these advantages in this detection techniques for PSA, this strategy was expected to hold considerable potential for point-of-care applications in clinical diagnostics.(2) In order to further extend the application range of the aforementioned strategy due to the high sensitivity and specifity of the PL-coated CNTs as the electrochemical labels for the immunoassay, we exploited single-stranded-DNA-wrapped CNTs as the electrochemical labels for the magnetic separation-based DNA hybridization assay in Chapter 3. In this method, the noncovalent wrapping of CNTs with single-stranded DNA via hydrophobic andπ-πstacking interactions not only imparts aqueous solubility and stability of CNTs but also allows a further molecular recognition reaction between the DNA onto the CNTs and the target DNA. Moreover, in order to enhance the discrimination capability for the single base mismatch, the magnetic beads were modified with the hairpin DNA. When the probes hybridized with the target nucleic acid, the allostery of the hairpin DNA on the MBs surface was switched and the ssDNA-wrapped CNTs labels were selectively collected by magnetic separation-based DNA hybridization reaction. With the assembly of these electrochemical labels on the C18H37SH-blocked electrode surface, a strong redox current was obtained. The developed strategy could quantitatively determine the target DNA in the dynamic range of 1 pM ~ 500 nM with the detection limit of 0.9 pM. The present approach has been demonstrated with the identification of a single base mutation in Hepatitis B virus specific sequence, and the wild type and mutant type were successfully scored.(3) Although the magnetic beads could enrichedly collect the electrochemical labels CNTs in the aforementioned strategies, these might increase the background current. Simultaneously, in order to further simplify the experiment procedure, we developed an electrochemical strategy based on strand displacement reaction coupling with the CNTs as electrochemical labels for the detection of the specific sequence DNA in Chapter 4. In this assay, the binding of the Watson-Crick base pair recognition event was stronger than the binding of the ssDNA and MWNTs, so the DNA hybridization between target DNA and the DNA probe wrapped on the nanotubes could actively remove DNA probe from the MWNTs surface and the precipitation of MWNTs adsorbed onto the 16-mercaptohexadecanoic-acid-modified Au electrode substantially mediated heterogeneous electron transfer between bare gold electrode and redox species in solution phase. In the absence of the target DNA, the MWNTs wrapped by ssDNA did not adsorbe on the 16-mercaptohexadecanoic-acid SAM due to strong electrostatic and hydration repulsions between the DNA-MWNTs and the negatively charged SAM in the neutral solution, thus reducing the background current. The experiment results demonstrated that the electrochemical signal of the electrode was correlated with the concentration of target DNA in the range from 10 pM ~ 100 nM with a detection limit of 5 pM.(4) In Chapter 5, coupling thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry with the CNTs as electrochemical labels, we developed a novel electrochemical strategy for the determination of Hg2+ in sewage. In this experiment, the effect of concentration of salt ion, temperature of strand displacement reaction and the interference of coexisting ions with Hg2+ on the performance of the assay was investigated. The experiment results indicated that the Hg2+ was determined in the range of 20 nM ~ 10μM with a detection limit of 10 nM.(5) Well-ordered aligned nanomaterials fabricated on the electrode not only provide plenty of active sites and a friendly microenvironment for the enzymes immobilization but also facilitate enzyme-substrate contact and improve the performance of the resulting biosensor. In Chapter 6, Ruthenium purple nanowire array (RPNWA) was synthesized using a polycarbonate (PC) membrane template via a direct electrodeposition technique on the glassy carbon electrode. The RPNWA-modified glass carbon electrodes as prepared were demonstrated to have high catalytic activity for the electrochemical reduction of hydrogen peroxide at low potential. Moreover, through the crosslinking of glucose oxidase on the nanoelectrode array surface, a biosensor for glucose was constructed. The expereiment results show that the biosensor displays rapid response and expanded linear response range besides excellent repeatability and stability.(6) The advantages of the layered nanomaterials are that the host has a layered structure with the adjustable interlayer distance and can be readily expanded to accommodate guest molecules of varying sizes. Also, the layered materials are suitable for the protein immobilization due to their excellent biocompatibility and chemical stability, large aspect ratio, high density surface charge, et al. In Chapter 7, we developed an electrochemical biosensor based on the immobilization of glucose oxidase (GOx) byα-zirconium phosphate (α-ZrP) layered nanomaterials and applied for the detection of glucose. In the present paper,α-ZrP layered nanomaterials were prepared by the method of direct precipitation and a facile protocol was described for immobilizing GOx in the interlayer regions of the layeredα-ZrP under ambient conditions at pH 7.0. Then, the GOx/α-ZrPs complex as prepared were dispersed in chitosan and immobilized on the glassy carbon electrode surface. Due to the open structure ofα-ZrP layered nanomaterials, this facilitated enzyme-substrate contact and improved the performance of the enzyme catalysis. The experiment results demonstrated that this biosensor could be used for the rapid and sensivitive determination of glucose, and the linear range was 0.01 mM～20.0 mM with the detection limit of 0.01 mM.(7) Small-molecule-linked DNA has emerged as a versatile tool for the interaction assay between small organic molecules and their protein receptors and has become a new method for the selection of drug and the detection of target proteins. This assay is based on translating the binding of small molecules to proteins into the presence of a specific DNA sequence, which enables us to probe the interaction between small organic molecules and their protein targets using various DNA sequence amplification and detection technologies. Consequently, this method can offer a promising platform for small molecule-protein interaction studies. Exonuclease I (Exo I) terminal protection assay is one of this method, which is based on our previous finding that single-stranded DNA (ssDNA) terminally tethered to a small molecule is protected from the degradation by exonuclease I (Exo I) when the small molecule moiety is bound to its protein. In Chapter 8, we developed terminal protection assay tested for a model streptavidin by the interaction between biotin and streptavidin. In this assay, when the biotin moiety at the 3'terminus of ssDNA interacted with the target protein streptavidin, the degradation of ssDNA was inhibited and this ssDNA then hybridized with the capture probe modified on the gold electrode. The ferrocene labels at the 5'terminus of this ssDNA were drawn close to the electrode surface, thus yielding a remarkable redox current. This strategy was demonstrated for quantitative analysis of the streptavidin and the quasi-linear ranger was 10 pM ~ 1 nM with desirable specificity and sensitivity.