A highly sensitive electrochemical carcinoembryonic antigen (CEA) immunosensor was fabricated by covalently immobilizing a monoclonal CEA antibody (anti-CEA, Ab(1)) and a mediator (thionine, Th) on a gold nanoparticle (AuNP)-encapsulated dendrimer (Den/AuNP). Multiwalled carbon nanotube (MWCNT)-supported secondary antibody (Ab(2))-conjugated multiple bienzymes, glucose oxidase (GOx), and horseradish peroxidase (HRP) (Ab(2)/MWCNT/GOx/HRP) were used as electrochemical labels. The highly sensitive detection was achieved by the increased HRP-electrocatalyzed reduction of hydrogen peroxide, which was locally generated by the enzyme GOx. The immunosensor surface was characterized using electrochemical impedance spectroscopy, atomic force microscopy, and quartz crystal microbalance techniques. The Den/AuNP and Ab(2)/MWCNT/GOx/HRP bioconjugates were characterized using high-resolution transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. Cyclic voltammetry and square wave voltammetry techniques were used to monitor the increased electrocatalyzed reduction of hydrogen peroxide by HRP. The linear dynamic range and the detection limit were determined to be 10.0 pg/mL to 50.0 ng/mL and 4.4 ± 0.1 pg/mL, respectively. The validity of the immunosensor response was tested in various CEA-spiked human serum samples, and the results were compared to those of an enzyme-linked immunosorbent assay method.

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Using gold nanoparticles (AuNPs) on “capillary enzyme-linked immunosorbent assay (ELISA)”, we produced highly sensitive and rapid assays, which are the major attributes for point-of-care applications. First, in order to understand the size effect of AuNPs, AuNPs of varying diameters (5 nm, 10 nm, 15 nm, 20 nm, 30 nm, and 50 nm) conjugated with Horseradish Peroxidase (HRP)-labeled anti-C reactive protein (antiCRP) (AuNP•antiCRP-HRP) were used for well-plate ELISA. AuNP of 10 nm produced the largest optical density, enabling detection of 0.1 ng/mL of CRP with only 30 s of incubation, in contrast to 10 ng/mL for the ELISA run in the absence of AuNP. Then, AuNP of 10 nm conjugated with antiCRP-HRP (AuNP•antiCRP-HRP) was used for “capillary ELISA” to detect as low as 0.1 ng/mL of CRP. Also, kinetic study on both 96-well plates and in a capillary tube using antiCRP-HRP or AuNP•antiCRP-HRP showed a synergistic effect between AuNP and the capillary system, in which the fastest assay was observed from the “AuNP capillary ELISA”, with its maximum absorbance reaching 2.5 min, while the slowest was the typical well-plate ELISA with its maximum absorbance reaching in 13.5 min.

[a] 1IntroductionAptamer basedb ioanalytical approaches with homogeneous and heterogeneous pathways represent promising tools for protein detection. Aptamers are synthetic DNA/ RNAb ased receptors synthesized from in vitro selection process [1][2][3].A ptamers with tridimensional conformation enables them to recognize biomolecules with affinities comparedt ot hoseo fa ntibodies [4][5][6][7].T hey can change conformation dependingo nt arget binding, which gives them great flexibility for designing novel biosensors with high sensitivitya nd selectivity.F abrication of biosensors with aptamers as biorecognitione lements were applied successfully and most of the reported works were related to thrombin (TB)-binding aptamer [8][9][10][11][12][13][14][15][16][17][18].T Bi s ap rotein that possesses many effectsi nc oagulation-related reactions responsible for blood clotting. It is as erine protease that converts soluble fibrinogen to insoluble strands of fibrin. It also plays av ital role in thrombosis and haemostasis and its detection is very usefulf or early diagnosis of intracerebral hemorrhage [19] and pulmonary metastasis [20].B ecause of clinical importance, ah ighly sensitive biosensor for the detection of TB would be of great importance.T Bd etection with aptamer based probe has severala dvantages in the area of biosensor [21].U ntil now,m any novel aptamer sensors based on fluorescence [22],s urface enhancedR aman spectroscopy [23],s urface plasmonr esonance [24],q uartz crystal microbalance [25] and electrochemistry [26,27] have been developed successfully.A mongt hem, electrochemical methods have been ap referable approachd ue to its high sensitivity,f ast response,l ow cost, and miniaturization. Although aptamer-based sensors (aptamer sensor) represent sensitive approaches,b ut the choice of am ore suitable aptamer immobilizing platformw ith sufficient biocompatibilitya nd conductivity is still ac hallenging task for the practicala pplication of aptamers ensors.Polydopamine (PD) is an ovel coating materials having extraordinary robust adhesion,w hich could be easilyd eposited all types of inorganic and organic surfaces with control film thickness and durable stability [28,29].A nother important advantage of this material is that it has ah igh biocompatibility [30].T hus,P Dc ould be an interesting material for the fabrication of ar obust electrochemical TB aptamers ensor. However, for an electrochemical aptamers ensor, the aptamer immobilizing platforms hould have sufficient conductivity in ordert of low the electron.S ince the conductivity of the PD film is low, thusi tn eeds to be increased for using as an aptameri mmobilizing platformi ne lectrochemical aptamer sensors. To increase the conductivity of the PD film, various nanomaterials can be usedfor the modificationo fthe film. Although, carbon nanotubes[ 10],q uantum dots [11],a nd gold nanoparticles (AuNPs) [12] were used for the fabrication of highly sensitiveT Ba ptamer sensors,h owever, the use of nanomaterials modified PD would be more advantag...