Development Of Amperometric Chemo-/biosensors Based On Thiol-ene Chemistry And Research On Highly Sensitive Piezoelectric Biosensing

The thiol-ene chemistry refers to the addition of a S-H bond across a double bond by either a free radical or ionic mechanism. Recently, the thiol-ene chemistry has already received extensive attention owing to its outstanding application potential in the fields of synthetic chemistry, material preparation and chemo-/biosensing. Because of their outstanding electrochemical and material characteristics, the polymer-modified electrodes have attracted substantial attention in various fundamental researches and practical applications. The conducting polymers (CPs) at their oxidized states of alkenes-like electron-conjugate structure can interact with thiols. In this dissertation, the reactions of thiols with CPs have been redefined as a researching branch of the emerging thiol-ene chemistry area, and a series of researches on material preparation and amperometric chemo-/biosensing are conducted based on thiol-ene chemistry of CPs.Electrochemical quartz crystal microbalance (EQCM) is a powerful and useful tool to study polymer-modified electrodes, e.g., the growth process of polymer, ions transport in polymer film. Compared with the conventional EQCM, the impedance-analysis-based QCM involved in this dissertation can be used to synchronously obtain multiple chemical/physical parameters and study material characteristics during an electrochemical perturbation. In this dissertation, the recent advances of thiol-ene chemistry, thiolated polymers, anodic stripping voltammetric determination of heavy metals, aptasensors as well as the applications of polymer-modified electrodes, QCM and surface plasmon resonance (SPR) are reviewed, and a series of amperometric chemo-/biosensing studies are conducted based on thiol-ene chemistry of CPs as well as the interaction of angiotensin converting enzyme and lisinopril studied by35MHz QCM and SPR, as summarized below.1. The electrochemical quartz crystal microbalance (EQCM) was used to study the electrosyntheses and electrochemical properties of two kinds of polyaniline (PANI)-thiol composite films in aqueous solutions, which were prepared by covalent binding of a thiol to the oxidized forms of PANI (PANIpost-thiol, protocol A), and electropolymerization of aniline in the presence of a thiol (PANIpoly-thiol, protocol B), respectively. The thiols involved were mercaptosuccinic acid (MSA), thioglycolic acid (TGA) and β-mercaptoethanol (ME). The PANIpost-thiol binding processes were monitored in situ with the EQCM, giving molar binding ratios (r, thiol vs. aniline unit of the polymer) of ca.0.50at saturation for these thiols. Both PANIpost-thiol and PANIpoly-thiol composite films from the carboxylated thiols showed controllable electroactivity of the PANI moiety in neutral even weakly alkaline phosphate buffer solutions (PBS), with maximum electroactivity roughly at r=0.11for PANIpost-MSA or at r=0.21for PANIpost-TGA. The PANI-thiol interaction was also supported by experiments of scanning electron microscopy, electrochemical surface plasmon resonance, Fourier transform infrared spectroscopy and ultraviolet-visible spectroscopy, and the interaction mechanism is briefly discussed. The PANIpost-thiol and PANIpoly-thiol composite films from the carboxylated thiols effectively electrocatalyzed the oxidation of ascorbic acid in pH=7.3PBS, and the PANIpost-thiol composite exhibited electrocatalytic activity higher than the relevant PANIpoly-thiol one under our experimental conditions.2. We report on the thiol-ene chemistry guided preparation of novel thiolated polymeric nanocomposite films of abundant anionic carboxylic groups for electrostatic enrichment and sensitive electroanalysis of cationic dopamine (DA) in neutral solution. Briefly, the thiol-ene nucleophilic reaction of a carboxylated thiol with oxidized polypyrrole (PPy) electrosynthesized on an Au electrode in the presence of solution-dispersed acidified multiwalled carbon nanotubes (MWCNTs) produced an a PPy-thiol-MWCNTs/Au electrode, and the PPy can be electrochemically overoxidized (OPPy) to form an OPPy-thiol-MWCNTs/Au electrode. The carboxylic groups of the polymeric nanocomposite film originate from the acidified MWCNTs, PPy-tethered carboxylated thiol, and OPPy. The carboxylated thiols examined are mercaptosuccinic acid (MSA) and thioglycolic acid (TGA), with β-mercaptoethanol (ME) as a control. Electrochemical quartz crystal microbalance, scanning electron microscopy, Fourier transform infrared spectroscopy and ultraviolet-visible spectroscopy were used for film characterization and process monitoring. Under the optimized condition, the differential pulse voltammetry (DPV) peak current response of DA oxidation at OPPy-MSA-MWCNTs/Au electrode is linear with DA concentration from1.00×10-9to2.87×10-6mol L-1, with a limit of detection of0.4nmol L-1, good anti-interferent ability and stability.3. We report on the thiol-ene chemistry guided preparation of a novel thiolated polymeric nanocomposite for sensitive differential pulse anodic stripping voltammetry (DPASV) determination of Cd2+and Pb2+on glassy carbon electrode (GCE). Briefly, the thiol-ene nucleophilic reaction of the functionalized thiol with oxidized polyaniline (PANI) synthesized in the presence of solution-dispersed acidified multiwalled carbon nanotubes (MWCNTs) yielded thiolated polymeric nanocomposite thiol-PANI/MWCNTs. The functionalized thiols examined include2,5-dimercapto-1,3,4-thiadiazole (DMcT),1,6-hexanedithiol and P-mercaptoethanol. The thiol-PANI binding processes were studied in situ with the quartz crystal microbalance, and the scanning electron microscopy, Fourier transform infrared spectroscopy and ultraviolet-visible spectroscopy were used to characterize the composite film properties. Under optimum conditions, a Bi/Nafion/DMcT-PANI/MWCNTs/GCE showed linear DPASV responses from0.02to20μg L-1for Cd2+and0.08to31μg L-1for Pb2+, with detection limits of0.01μg L-1for Cd2+and0.04μg L-1for Pb2+(S/N=3), respectively, as well as good stability and reproducibility. The electrode was also used to determine Cd2+and Pb2+in real water samples with satisfactory recovery.4. We report on a novel electrochemical aptasensor based on the thiol-ene chemistry of thiolated thrombin aptamer (TTA), polyaniline (PANI) and its multiwalled carbon nanotubes (MWCNTs) composite. The thiol-ene chemistry guided preparation of thiolated polymeric bionanocomposite (TTA-PANI/MWCNTs) modified glassy carbon electrode exhibits good electroactivity of self-doped PANI, after the TTA-thrombin interaction, the DPV current response decreases. The resultant aptasensor has a detection limit of thrombin down to0.3pM (S/N=3). The scanning electron microscopy, cyclic voltammetry and ultraviolet-visible spectroscopy were used to characterize the film properties.5. We report on a novel and highly sensitive electrochemical aptasensor based on the thiol-ene chemistry of1,6-hexanedithiol (HDT), p-benzoquinone (BQ) and thiolated thrombin aptamer (TTA) as well as self-assembled monolayer technique. The sandwich-type TTA-BQ-HDT modified gold electrode prepared by thiol-ene chemistry shows good electroactivity of BQ/HQ, and after the TTA-thrombin interaction, the DPV current response decreses. The resultant aptasensor has a detection limit of thrombin down to20fM (S/N=3).6. Angiotensin converting enzyme (ACE) plays a pivotal role in blood pressure regulation, and its interaction with an ACE inhibitor (ACEI) is an important research topic for treatment of hypertension. Herein, a low reagent consumption, multiparameter and highly sensitive quartz crystal microbalance (QCM) at35-MHz fundamental frequency was utilized to monitor in situ the binding process of solution lisinopril (LIS, a carboxylic third-generation ACEI) to ACE adsorbed at a1-dodecanethiol (C12SH)-modified Au electrode. From the QCM data, the binding molar ratio (r) of LIS to adsorbed ACE was estimated to be2.3:1, and the binding and dissociation rate constants (k1and k-1) and the binding equilibrium constant (Ka) were estimated to be k1=4.1×106L mol-1s-1, k-1=7.3×10-3s-1and Ka=5.62×108L mol-1, respectively. Comparable qualitative and quantitative results were also obtained from separate experiments of cyclic voltammetry, electrochemical impedance spectroscopy and surface plasmon resonance measurements.