Au nanoparticle decorated graphene nanosheets for electrochemical immunosensing of p53 antibodies for cancer prognosis.

The accurate quantification of the level of p53 antibodies in serum is crucial for cancer prognosis. We report a novel and sensitive label-free immunosensor based on gold nanoparticles (Au NPs) self-assembled onto electrochemically reduced graphene oxide (ERGO) for the detection of p53 antibodies. An electrografted p-aminophenol organic layer was used to immobilize graphene oxide (GO) onto the surface of screen printed carbon electrodes (SPCE). The Au NP/ERGO hybrid interface provides a large surface area for the effective immobilization of p53 antigens, as well as it ascertains the bioactivity and stability of immobilized p53 antigens. Scanning electron microscope, Raman and X-ray photoelectron spectroscopies were used to monitor the sensor fabrication and cyclic voltammetry was used to quantify the extent of Au NPs' surface coverage by p53 antigens. Square wave voltammetry (SWV) of a [Fe(CN)6](3-/4-) couple was employed to investigate the immunosensor fabrication and to monitor the binding events between p53 antigens and p53 antibodies. Under optimized experimental conditions, the biosensor displayed good sensitivity and specificity. The p53 antibodies were detected in a concentration as low as 0.088 pg mL(-1) with a linear range from 0.1 pg mL(-1) to 10 ng mL(-1). The high sensitivity of the immunosensor may derive from the high loading of p53 antibodies on Au NPs which increases the number of binding events.

DNA aptamers selection and characterization for development of label-free impedimetric aptasensor for neurotoxin anatoxin-a.

High affinity DNA aptamers against anatoxin-a (ATX), the smallest potent neurotoxin (Mol. Wt, 165.23 Da) were selected and identified in vitro using the systematic evolution of ligands by exponential enrichment (SELEX) approach. Aptamers with dissociation constants (Kd) of nanomolar range were isolated. The aptamer sequence of highest affinity was used to design a label-free impedance based aptasensor to assay ATX, for which there are no reported biosensors so far. The aptamer self assembled monolayer is formed on a gold electrode using the disulfide modified aptamer. The assembly process of the aptasensor was characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Upon ATX binding to the immobilized aptamer, a significant decrease in the electron-transfer resistance was observed as a result of the aptamer conformation change, which is used as the sensor signal. The aptasensor showed a limit of detection of 0.5 nM and a wide linear range for ATX concentrations between 1 nM and 100 nM. The Kd of anti-ATX aptamer was calculated by electrochemical methods as well as the fluorescence. Interestingly, the Kd that was calculated from the aptasensor signal showed a lower value implying that the anchoring of the aptamer on the Au surface enhanced its affinity to ATX. The ATX aptasensor showed high stability as well as high specificity against common cynaobacterial toxins. Further development of biosensors that use anatoxin-a binding aptamers as a new recognition receptors could provide potential alternatives to the traditional assays for fast and simple monitoring of anatoxin-a.

In vitro selection, characterization, and biosensing application of high-affinity cylindrospermopsin-targeting aptamers.

Contamination of freshwater with cyanotoxin cylindrospermopsin (CYN) represents a significant global concern for public health. The sensitive detection of CYN is necessary to effectively manage and control the treatment of water resources. Here we report a novel, highly sensitive label-free aptasensor for CYN analysis, using aptamers as specific receptors. We have selected the DNA aptamers from a diverse random library using the in vitro screening SELEX approach. The aptamers exhibited high affinity for CYN with Kd of nanomolar range. One aptamer exhibited conformational change upon CYN recognition (CD analysis) and was used to fabricate the label-free impedimetric aptasensor for CYN. A self-assembled monolayer from a disulfide-derivatized aptamer was formed on a gold electrode to fabricate the aptasensor. Upon CYN capturing to the aptasensor surface, a marked drop in the electron transfer resistance was obtained, which was used as the principle of detection of CYN. This resulted from the aptamer's conformational change induced by CYN recognition. The present aptasensor could detect CYN with the limit of detection as low as 100 pM and a wide linear range of 0.1 to 80 nM. When mounted on the gold surface, the aptamer exhibited a lower dissociation constant for CYN than that observed in the fluorescence assay, implying that the anchoring of the aptamer on the Au surface improved its affinity to CYN. Moreover, the aptasensor showed high specificity toward other coexistent cyanobacterial toxins of microcystin-LR and Anatoxin-a. Further biosensor designs will be generated using those aptamers for simple and sensitive CYN monitoring.

Electrochemical impedance immunosensor based on gold nanoparticles-protein G for the detection of cancer marker epidermal growth factor receptor in human plasma and brain tissue.

A sensitive label-free impedimetric immunosensor for the detection of cancer biomarker epidermal growth factor receptor (EGFR) was developed with a limit of detection as low as 0.34 pg mL(-1) in PBS and 0.88 pg mL(-1) in human plasma. The gold nanoparticles were electrodeposited to modify the gold surface and to increase the electrochemical active area by a factor of approximately 3, i.e. by 68%. Protein G was used as scaffold for well oriented EGFR antibodies immobilization. Under optimal experimental parameters, the impedance changes were used for the detection of EGFR with a wide dynamic range of 1 pg mL(-1)-1 µg mL(-1). The immunosensor showed an excellent reproducibility and selectivity against biomarkers, murine double minute 2 and platelet derived growth factor receptor. The excellent analytical performance of the EGFR immunosensor in terms of selectivity, sensitivity and low detection limit might be attributed to the synergetic effect between the Au nanoparticles and the protein G scaffold. The matrix effect from mouse brain tissue homogenate was also studied and the immunosensor showed excellent recoveries ranging from 98.3% to 115% and RSD of 1.55-6.17. Finally, our developed strategy could open new avenues for clinical screening and prognosis of tumors.

Label-free impedimetric immunosensor for ultrasensitive detection of cancer marker Murine double minute 2 in brain tissue.

The detection of cancer biomarkers is as important tool for the diagnosis and prognosis of cancer such as brain cancer. Murine double minute 2 (MDM2) has been widely studied as prognostic marker for brain tumor. Here we describe development of a new sensitive label free impedimetric immunosensor for the detection of MDM2 based on cysteamine self assembled monolayers on a clean polycrystalline Au electrode surface. The amine-modified electrodes were further functionalized with antibody using homobifunctional 1,4-phenylene diisothiocyanate (PDITC) linker. The assembly processes of the immunosensor had been monitored with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques using Fe(CN)(6)(3-/4-) solution as redox probe. The impedance changes upon binding of MDM2 protein to the sensor surface was utilized for the detection of MDM2. The increase in relative electron-transfer resistance (ΔR/R(0)%) values was linearly proportional to the concentration of tumor marker MDM2 in the wide dynamic range of 1pg/ml-1µg/ml. The limit of detection was 0.29pg/ml in phosphate buffer saline (PBS) and 1.3pg/ml in mouse brain tissue homogenate, respectively. The immunosensor showed a good performance in comparison with ELISA for the analysis of the MDM2 in the cancerous mouse brain tissue homogenates. Moreover, the immunosensor had a good selectivity against epidermal growth factor receptor (EGFR) protein, long-storage stability and reproducibility. It might be become a promising assay for clinical diagnosis and early detection of tumors.