| Electrogenerated chemiluminescence (ECL) is a special form of chemiluminescence in which light emission is triggered from electrochemical reactions. Due to the combination of electrochemical and luminescent techniques, the ECL technique exhibits simplified manipulation, high sensitivity and wide dynamic response range, and has become an important and valuable tool in analytical chemistry. In this study, ECL cholesterol and concanavalin A biosensors were constructed based nanomaterials and enzyme. Our proposed strategy would extend the application of ECL technique in enzyme and biospecific interaction-based biosensors. The main work was listed as follows.Part 1 Bi-pseudoenzyme synergetic catalysis to generate a coreactant of peroxydisulfate for an ultrasensitive electrochemiluminescence-based cholesterol biosensorA novel electrochemiluminescence (ECL) enzyme biosensor for the ultrasensitive detection of cholesterol was designed based on a bi-pseudoenzymatic reaction to generate a coreactant of peroxydisulfate for signal amplification. In this work, hemin functionalized graphene (hemin-GR) was synthesized and used to immobilize cholesterol oxidase (COx) to construct an ECL biosensor for cholesterol. When cholesterol was added to the detection solution, COx catalyzed the oxidation of cholesterol to generate H2O2, which could be further catalyzed by hemin to produce O2 as the coreactant in the peroxydisulfate solution system for signal amplification. The linear range for cholesterol detection was 3.3-1.5 × 103 nmol·L-1, with a lower detection limit of 1.0 nmol·L-1 (signal to noise ratio=3). Therefore, the detection limit and sensitivity of the biosensor were improved. This novel strategy offers the advantages of simplicity, improved sensitivity, good selectivity, and repeatability, thus holds a promise for use in sensitive bioassays for clinical determination of cholesterol levels.Part 2 An ultrasensitive electrochemiluminescent biosensor for the detection of concanavalin A based on poly(ethylenimine) reduced graphene oxide and hollow gold nanoparticlesA highly sensitive electrochemiluminescent (ECL) biosensor was designed for the detection of concanavalin A (ConA) based on glucose oxidase (GOx) as a recognition element by carbohydrate-lectin biospecific interaction, and poly(ethylenimine) (PEI) reduced graphene oxide and hollow gold nanoparticles (HAuNPs) as supporting matrix and signal amplifier. The modification process and detection principle of the biosensor are briefly described as follows. First, PEI reduced graphene oxide with abundant amino groups was cast onto the surface of glassy carbon electrode to adsorb HAuNPs for improving the signal intensity in luminol/H2O2 ECL system. Next, GOx was further assembled onto the electrode. In the presence of glucose in the detection solution, GOx catalyzed glucose to generate H2O2 in situ, which served as a co-reactant of luminol to enhance the ECL signal of luminol. Based on the fact that ConA could result in a decrease in ECL signal when immobilized on the electrode, an ECL biosensor was prepared for the determination of ConA. The ECL signal intensity was linear with the logarithm of ConA concentration and the linear range was from 1.0 to 20 ng·mL-1 with a low detection limit of 0.31 ng·mL-1 (signal to noise ratio=3). This strategy led to a nearly 1000-fold improvement in detection limit for ConA assays compared with previously reported method, thus exhibiting a great potential application in sensitive bioassays of ConA.Part 3 A sandwich-like electrochemiluminescent biosensor for the detection of concanavalin A based on a C60-educed graphene oxide nanocomposite and glucose oxidase functionalized hollow gold nanospheresA sensitive sandwich-like electrochemiluminescent (ECL) biosensor was designed for the detection of concanavalin A (ConA) using a C60-reduced graphene oxide (C60-rGO) nanocomposite as a platform and glucose oxidase (GOx) decorated hollow gold nanospheres (HAuNPs) as a label. First, C60-rGO with a large surface area was prepared for combining with phenoxy-derivatized dextran, which served as the recognition element for interacting with ConA by biospecific carbohydrate-protein (lectin) interactions. Then, GOx decorated HAuNPs (GOx-HAuNPs) were linked to the electrode surface through the biospecific interaction between the intrinsic carbohydrate residues of GOx and ConA. These localized GOx and HAuNPs amplified the ECL signal of luminol intensely, which was achieved by the efficient catalysis of the GOx towards the oxidation of glucose to in situ generate an improved amount of hydrogen peroxide (H2O2) as a coreactant, and the excellent catalysis of HAuNPs towards the ECL reaction of luminol/H2O2. The prepared biosensor exhibited a sensitive response for the determination of ConA, ranging from 0.10 to 100 ng·mL-1 with a detection limit down to 30 ng·mL-1 (signal to noise=3). With excellent stability, sensitivity, selectivity and simplicity, the prepared biosensor showed great prospects in lectin sensing or carbohydrate sensing. |
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