| As a major component of life, proteins have played an important role in entire lifeactivities and regulations. Therefore, Detection of proteins expression particular in diseasestate will help us to better understand of the life and to prevent, diagnosis and treatment ofdiseases significantly.Biosensor based on biological molecules as recognition elements, becomes the mostpromising methods for protein detection with its high specificity, high sensitivity, andconvenient operation. Currently antibodies are the most widely used biomolecularrecognition element, but its further application on biosensors was limited because of theirlong preparation period, instability, Stringent environmental requirements and othershortcomings. In recent years, more and more new recognition elements are introduced intothe preparation of biosensor, wherein the nucleic acid aptamer is the most remarkable one.Aptamers are single-stranded DNA or RNA screened from a large single nucleotide libraryby “Systemic Evolution of Ligands by Exponential Enrichment technique”(SELEX). Inaddition to their high affinity binding ability and high specific recognition characteristicscompare with antibodies, they also exhibit many other distinctive advantages such assmaller molecular weight, wide range of target, accurate synthesis, ease of labeling，stability and can be refolding after denatured, etc. Therefore aptamer is becoming a newideal biosensor recognition element. Electrochemical biosensors are a class of detectionsensor which convert biological concentration of the substance into a detectable signal byrecognition element with characters of high sensitivity, without affected by color of thesolution, quickly response, convenient operation, inexpensive, portable, etc., Combinedwith the aptamer, electrochemical aptasensors become a new hotspot beyond research ofimmune electrochemical sensors and DNA electrochemical sensors.This project aims to establish a high-sensitivity, high specificity, and convenientoperation electrochemical aptamer biosensor for protein detectation. We set the aptamer as highly specific molecular recognition element of biosensor， and construction highsensitivity, high specificity of label free aptamers electrochemical sensors based onenzyme-based amplification strategies for protein detection. And study the application ofaptasensor in platelet growth factors (PDGF) and snake venom detection. Works containthe following sections:1. Review section. After general introducing of principle of SELEX and theadvateges of aptamer, the electrochemical sensor and its applications are highlighted.Moreover, the strategy of label-free base on enzyme-aplification in electrochemicalaptasensor was presented. And at the same time, this paper introduces the significance ofprotein detection and the present development of protein detection methodology2. Construction of label free electrochemical aptasensor base on enzyme-basedamplification strategies. Recently, there has been substantial attention in the directelectron transfer (DET) of many redox enzymes. However, DET of most enzymes on baresolid electrodes is difficult to achieve because of the instability of matrix and the deeplyembedded active sites in enzyme molecules. In order to overcome this drawback, greateffort has been made to develop novel reagentless biosensor devices based on DET ofenzyme by immobilizing it on nanomaterials modified electrode. Thereinto, graphene (Gra)as an ideal base has been widely applied due to the unique physical and chemical properties,such as high surface area, excellent conductivity and ease of functionalization. On the otherhand, gold nanoparticles (GNPs) are considered as satisfactory nanomaterials because oftheir excellent biocompatibility, strong adsorption ability and good conductivity. Suchproperties make poly (diallyldimethylammonium chloride)(PDDA)-protected Gra-GNPs(P-Gra-GNPs) composite be more attractive as a host immobilization matrix, which notonly assist DET between active sites of enzyme and electrode, but also provide a favorablemicroenvironment for immobilizing enzymes. For this proposed aptasensor, poly(diallyldimethylammonium chloride)(PDDA)-protected graphene-gold nanoparticles(P-Gra-GNPs) composite was firstly coated on the glassy carbon electrode surface to formthe interface of biocompatibility and huge surface area for the adsorption of GOD layer.Subsequently, gold nanoclusters (GNCs) was deposited on the surface of GOD to capturePDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to blockthe remaining active sites of the GNCs and avoid the nonspecific adsorption. Herein, based on the above observations, we first developed a novel reagentless and mediatorlesselectrochemical aptasensor based on the DET of glucose oxidase (GOD) for the detection ofplatelet-derived growth factor (PDGF).3. Detection of PDGF based on electrochemical aptasensor. Platelet-derivedgrowth factor (PDGF) is a small molecular weight mitogenic peptides regulating factor,which named with its presentation in platelet α particles in normal physiological conditions.PDGF will be released from platelets when blood coagulation, and be activated to stimulatefibroblasts, glial cells, and smooth muscle cells from G0/G1phase into the proliferation cycle.Detection of platelet-derived growth factor plays an important role in fibrosis, cancerdiagnosis and prognostic evaluation. In this work, a new label-free electrochemicalaptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growthfactor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). With thedirect electron transfer of double layer GOD membranes, the aptasensor showed excellentelectrochemical response and the peak current decreased linearly with increasing logarithm ofPDGF concentration from0.005nM to60nM with a relatively low limit of detection of1.7pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-termstability, which provided a new promising technique for aptamer-based protein detection.4. Screening of Snake venom species-specific aptamer. Snakebite diagnosisrequires accurate identification of the type of snake venom. Current laboratory diagnosis ofsnakebite venom relies mainly on immunological diagnostic methods based on antibodies.But its poor specificity, complex preparation process limit futher application. The principleof aptamer selection (SELEX) inspires us that we can screen target species-specific aptamerfrom ssDNA library by introducing several deplete-screening processs for depleting crosstargets. We call this technology as deplete systematic evolution of ligands by exponentialenrichment (Deplete-SELEX). Using of Deplete-SELEX we obtain Naja naja atra venomspecies-specific nucleic acid aptamers to identify Agkistrodon acutus Guenther venom,Agkistrodon halysvenom, and Trimeresurus mucrosquamatus Canto venom to takeadvantage of subsequent establishment of aptamer-based electrochemical biosensors forsnak venom identification.5. Identification of snake venom by electrochemical aptasensor. Currentlysnakebite diagnosis relies on clinical symptoms (such as wound, local signs, etc.), routine laboratory tests (hemoglobin, urine bilirubin, etc.) or confirms the species of captured snake.But in the early clinical symptoms of snakebites are often not obvious, and even because ofthe majority of snakebites occur in the evening or in the mountains it is difficult to identifysnake species, which leading to false diagnosis, and miss guidance of the use of antivenintreatment. Over the years, methods such as immuno electrophoresis, ELISA and RIA areused to detect species-specific snake venom components from patient specimens bite byvarious snakes. Affected by material types, diagnostic antibodies quality and other factorsthese methods show low sensitive. Electrochemical aptamer biosensor with high sensitivity,specificity and portable characters will become ideal venom rapid detection means. Bycombining venom species-specificity aptamers with electrochemical biosensor we constructan electrochemical aptamer biosensor for snake venom detection. The sensor shows goodspecificity and sensitivity in snake venom identification, and may become an importantmeans for venom detection.In summary, we have successfully fabricated a highly sensitive and selectiveaptasensor for PDGF and snake venom detection by using the direct electrochemistry ofGOD on P-Gra-GNPs composite matrix. Moreover, the proposed method holds greatpromise for other proteins or biomarkers detection. Meanwhile we have establishedDeplete-SELEX screening technology, and successfully obtained Naja naja atra venomspecies-specific aptamers. The advantages of the proposed aptasensor were shown asfollows: Firstly, the P-Gra-GNPs composite film possessed large specific surface area andexcellent biocompatibility, which not only increased the immobilization of GOD, but alsoretained the active of immobilized biomolecules and enhanced the stability of aptasensor.Secondly, the GNCs film provided a stable and porous surface for PBA immobilization andfurther amplified the electrochemical signal. In addition, the double layer GOD membranesas tracer brought about higher current response and higher sensitivity. On the basis of theabove reasons, the proposed aptasensor showed relatively low detection limit, satisfactoryselectivity, acceptable stability and reproducibility. |
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