| During the past several decades, we have witnessed the fast development in industry and technology. However, the pollution induced during the process is also severe. Many technical methods have been constructed to detect the pollutants. However, a large portion of them is sophisticated instruments which are difficult for daily usage and need long detection period due to instrument modulation. To make daily detection simple and convenient, the sensors for daily detection usage are in great need. During my phd studies, I mainly concentrate on the development of very simple, easy handling methods for detection and any one without training can conduct those experiments according to the developed protocols.The first part is on the development of saxitoxin (STX) detection method. Saxitoxin is a natural neurotoxin which can block the sodium channel inducing paralysis even death. It is often caused by the ingestion of shellfish with accumulated toxins from phytoplankton species. The toxin is nontoxic to shellfish due to the less importance of sodium channel in shellfish body. However, due to its high stability towards heat, acid and oxidation, this guanidinium based toxin is often difficult to get rid of by simple baking. Therefore, it is in great necessity that the toxin is detected before it is digested into human body. Seven methods have been applied for STX detection. The sodium channel based method is constraint due to the difficulty in extracting the sodium channel from biological bodies and this extraction procedure needs long time training in expertise. The antibody based detection method offers high specificity but the protein is less stable under non-biological conditions and is easily affected by environmental changes. The animal based method like the usage of mouse is not only under ethic issues but also individual differences and the HPLC post-column fluorescence method is accurate but the instrument needs training and long-time usage. Herein, we developed a method for saxitoxin detection based on DNA conformational changes using a commonly used dye crystal violet (CV) as reporter. Guanine-rich DNA is DNA strands with guanine bases. Four guanine bases can interact through hydrogen bonding to form a planar G-quartet. The hydrophobic stacking of those G quartets stabilized by a monovalent cation induces the formation of G-quadruplex. Since the saxitoxin has two guanidinium backbones which are similar with guanine in structure, we speculate that the addition of saxitioxn may interfere with the formation of cation induced G-quadruplex formation. In our study, we used one widely studied G-quadruplex sequence-thrombin binding aptamer (TBA) which has two layers of G-quartets and15bases. Using potassium ions as G-quadruplex formation inducer, we found that the addition of saxitoxin can reduce the formation of G-quadruplex and a linear relationship can be extrapolated using circular dichroism (CD) signal versus saxitoxin concentration. Since TBA is a very short sequence, the fluorescence change before and after TBA G-quadruplex formation is small, therefore, the inserted dye detection method was used to reflect the conformational changes. Crystal violet (CV) is an organic molecule and the hydrophobic interaction makes the molecules tend to combine with random coiled DNA so that the fluorescence is on. However, when metal ions exist, CV cannot expel ions from the cavity, therefore, the fluorescence is weak. With the addition of STX, the G-quadruplex becomes opened and due to the electrostatic interaction between the CV and DNA strand, the CV turns on. Therefore, a turn-on sensor has been constructed using TBA as a sensing element and CV as a reporter part. However, the CV based insertion dye detection method has a shortcoming in that the detection limit is usually too high for daily detection.Part two is the construction of biosensors based on quartz crystal microbalance-dissipation (QCM-D). QCM-D is a powerful tool for in-situ studies of interface interaction and label-free, real-time characterization and quantification of reaction mechanism because it can not only reflect the reaction in the interfaces the mass changes but also viscoelasticity character of the adsorbed layer. However, few efforts are concentrated on the development of QCM-D for sensing platform. Therefore, during my phd studies, I mainly concentrate on the development of QCM-D based sensing platform constructions. The first one is for small molecule detection. Since the frequency signal is proportional to mass change, the higher molecular weight the substance, the higher frequency making the detection of large molecular weight substances detection easier. However, to detect small molecular weight molecules is needed to explore in order to expand the application of QCM-D. In my work, I use gold nanoparticles (AuNPs) which have large mass and easy modification characteristics to enhance the detection resolution and lower the detection limit. The QCM-D crystal was first modified with a layer of immobilization DNA using mercapto-gold interaction and then through base pair interaction, the linker part containing sequence for adenosine detection was hybridized onto the chip. If adenosine contains in the sample, the adenosine sequence can capture the target hampering the further hybridization of reporter DNA loaded with AuNPs and induces large signal changes due to the AuNPs. With the enhancement of AuNPs, the detection limit is enhanced at least3orders of magnitude and the resolution is higher since the signal range lies in100Hz instead of1Hz which is difficult to differentiate between signal and noise.Another work uses QCM-D is to explore the adsorption of microcystein onto polymer modified QCM-D chips. The commonly studied adsorption substrate is activated carbon. However, the functional groups’influences on the adsorption are unknown for the difficulty in modification. Therefore, we found one substrate substituting the existing widely studied activated carbon with easy changing functional groups and convenient obtainable ways-the polystyrene based polymers. Many studies on adsorption are detected by detecting the mobile phase analyte concentration change to plot the adsorption kinetics. With the aid of QCM-D, we are able to monitor the kinetics in real time. Moreover, with the analysis of dissipation, the adsorption mechanism can be inferred and amine modified polystyrene in neutral solution is recommended for adsorption of microcystein.The third part is about the construction of sensors based on gold nanoparticles (AuNPs) colorimetric effects. AuNPs have surface plasmon resonance (SPR) effects which make them change color when the distances between particles changes. When they aggregate, the color changes from red to purple or even blue, gray, depending on the aggregation extent. Based on this phenomenon, a simple pH alarm was constructed using DNA as sensing element and AuNPs as reporter part. It is found that adenine base can base paired with each other under acidic conditions. When the pH is low, sequences full of adenines can base pair to form duplex while at high pH condition, the sequence remain in single stranded form. In the AuNPs system, DNA single strands have bare bases that can interact with AuNPs through nitrogen-AuNPs coordination. However, when formed in duplex, the base pair interaction is substituted by hydrogen bonding among DNA strands. Without the protection of DNA, AuNPs is susceptible to aggregation under high salt concentration. From the color change, the pH can be easily differentiated. Moreover, by changing the inherent influences, the detection range can be modulated. |
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