Interactions of DNA with graphene and sensing applications of graphene field-effect transistor devices: A review

Graphene field-effect transistors (GFET) have emerged as powerful detection platforms enabled by the advent of chemical vapor deposition (CVD) production of the unique atomically thin 2D material on a large scale. DNA aptamers, short target-specific oligonucleotides, are excellent sensor moieties for GFETs due to their strong affinity to graphene, relatively short chain-length, selectivity, and a high degree of analyte variability. However, the interaction between DNA and graphene is not fully understood, leading to questions about the structure of surface-bound DNA, including the morphology of DNA nanostructures and the nature of the electronic response seen from analyte binding. This review critically evaluates recent insights into the nature of the DNA graphene interaction and its affect on sensor viability for DNA, small molecules, and proteins with respect to previously established sensing methods. We first discuss the sorption of DNA to graphene to introduce the interactions and forces acting in DNA based GFET devices and how these forces can potentially affect the performance of increasingly popular DNA aptamers and even future DNA nanostructures as sensor substrates. Next, we discuss the novel use of GFETs to detect DNA and the underlying electronic phenomena that are typically used as benchmarks for characterizing the analyte response of these devices. Finally, we address the use of DNA aptamers to increase the selectivity of GFET sensors for small molecules and proteins and compare them with other, state of the art, detection methods.     

Ultra-sensitive immunosensor for detection of hepatitis B surface antigen using multi-functionalized gold nanoparticles

The signal amplification for analytical purposes has considerable potential in detecting trace levels of analytes for clinical, security or environmental applications. In the present report a strategy based on a sandwich type immunoassay system was designed for the detection of hepatitis B surface antigen which exploits the specific affinity interaction between streptavidin and biotin recognition systems. The method involves the specific coupling of multi-functionalized gold nanoparticles (bearing biotin and luminol molecules) to the streptavidin modified by secondary antibody. The chemiluminescent signal is produced by the gold nanoparticles in the presence of HAuCl₄ as catalyst and hydrogen peroxide as oxidant. The immunosensor was able to detect hepatitis B surface antigen in the linear concentration range from 1.7 to 1920 pg mL⁻¹ and the detection limit of 0.358 pg mL⁻¹, at signal/noise = 3.     

Electro‐nano Diagnostic Platforms for Simultaneous Detection of Multiple Cancer Biomarkers

In this study, a bimetallic nanomaterial‐based electrochemical immunosensor was developed for the detection of carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF) cancer biomarkers at the same time. CEA and VEGF biomarkers are indicators for colon and breast cancers and stomach cancers, respectively. During the study, gold nanoparticle (AuNp), lead nanoparticle (PbNp), copper nanoparticle (CuNp) and magnetic gamma iron(III)oxide (γFe₂O₃ Np) were synthesized, characterized and used together for the first time in the structure of an electrochemical biosensor based on anti‐CEA and anti‐VEGF. For this purpose, Au SPE based sandwich immunosensor was fabricated by using labeled anti‐CEA (labeled with Pb⁺²) and labeled anti‐VEGF (labeled with Cu⁺²). As a result, CEA and VEGF biomarkers were detected following the oxidation peaks of label metals (Pb⁺² and Cu⁺²) by using differential pulse voltammetry. After the experimental parameters were optimized, the linear range was found in the concentration range between 25 ng/mL and 600 ng/mL with the relative standard deviation (RSD) value of (n=3 for 600 ng/mL) 3.33 % and limit of detection (LOD) value of 4.31 ng/mL for CEA biomarker. On the other hand, the linear range was found in the concentration range between 0.2 ng/mL and 12.5 ng/mL with the RSD value of (n=3 for 12.5 ng/mL) 5.31 % and LOD value of 0.014 ng/mL for VEGF biomarker. Lastly, sample application studies for synthetic plasma sample and interference studies with dopamine, ascorbic acid, BSA, cysteine and IgG were carried out.     

A Prostate Specific Antigen Immunosensor Based on Biotinylated‐Antibody/Cyclodextrin Inclusion Complex: Fabrication and Electrochemical Studies

Immobilization of biomolecules with a proper orientation is considered as a basis for diverse biotechnological applications. Herein, we report a host‐guest inclusion complexation between β‐cyclodextrin (β‐CD) and biotin as a versatile approach for the immobilization of biomolecules. As a practical application, a sandwich‐type electrochemical immunosensor was designed for the determination of prostate specific antigen (PSA). The immunosensor was fabricated by in situ electropolymerization of poly(N‐acetylaniline) onto a rGO‐modified Pt electrode. Then, β‐CD was covalently grafted onto the over‐oxidized polymer backbone. For improving the efficiency of the assay, AuNPs were casted on the polymeric film, on the surface of which thionine (TH) as an electron mediator was covalently immobilized. Using a host‐guest inclusion complexation between β‐CD and biotin, a β‐CD/biotin‐Ab₁/PSA/Ab₂‐horseradish peroxidase (HRP) sandwich was formed on the electrode surface. The analytical signal was produced via electrochemical reduction of TH_ox, generated by biocatalytic oxidation of the TH_red in the presence of HRP/H₂O₂. Under optimal conditions, the proposed sensor responded linearly to PSA in the range from 10.0 pg mL⁻¹ to 25.0 ng mL⁻¹, with a low detection limit of 6.7 pg mL⁻¹ (S/N=3). Kinetic parameters of the interaction of β‐CD with Ab₁ were also investigated. Finally, the applicability of the immunosensor was successfully investigated for the detection of PSA in human serum samples.     

A novel method for the sensitive detection of mutant proteins using a covalent-bonding tube-based proximity ligation assay

Tumorigenesis is the cumulative result of multiple gene mutations. The mutant proteins that are expressed by mutant genes in cancer cells are secreted into the blood and are useful biomarkers for the early diagnosis of cancer. However, some difficulties exist; for example, the same gene will express different protein mutants in different patients, and early tumors secrete only small amounts of mutant protein. Thus, the presence of mutant proteins in plasma has not previously been exploited for the early diagnosis of cancer. Proximity ligation assay is a protein-detection method that has been developed in recent years and has been widely used because of its high sensitivity. However, this approach still suffers from some shortcomings that should be addressed. In this paper, we develop a covalent-bonding tube-based proximity ligation assay (TB-PLA). The limit of detection of TB-PLA for 0.001 pM, and the method exhibited a broad dynamic range of up to seven orders of magnitude. Furthermore, we coupled the conformation-specific antibody PAb240 of p53 mutants to PCR tubes for TB-PLA. The assay was capable of detecting an approximately 500-fold lower concentration of mutant p53 in serum compared with sandwich ELISA. Thus, we demonstrate TB-PLA to be a highly sensitive and effective approach that is suitable for the early clinical diagnosis of cancer using the conformation-specific antibodies of protein mutants.     

Immuno-fluorescence based Vi capsular polysaccharide detection for specific recognition of Salmonella enterica serovar Typhi in clinical samples

Typhoid fever is a life threatening bacterial infection that remains a major global health concern. This continued high burden associated with significant morbidity and mortality rate demands specific and rapid detection technique. This work reports a new sandwich type fluorescence immunoassay format using polymyxin B, a cationic receptor molecule, as a binder agent while anti-Vi antibody served as the capturing agent for specifically detecting Salmonella enterica serovar Typhi. Anti-Vi IgG antibody raised against Vi–BSA conjugate revealed affinity of 7.779 nM⁻¹ signifying immunodominancy of O-acetyls groups in Vi polysaccharide. The detection limit of the developed assay was around 10¹ cells mL⁻¹ of Vi expressing Salmonella enterica serovar Typhi with a correlation coefficient (R²) equal to 0.97. Positive response obtained for all the tested serovar Typhi clinical isolates as well as the pathogen spiked blood samples recommended specificity and accuracy of Vi antigen as a biomarker during typhoid fever. The intra- and inter-assay precision with Vi spiked samples were satisfactory revealing coefficient of variance (CV%) with a mean of 4.05% and 5.97% respectively. This may be the novel attempt and constructive report on the fluorescence based detection of Vi antigen of serovar Typhi in the epidemic as well as pandemic outbreaks.     

Selective capturing and detection of Salmonella typhi on polycarbonate membrane using bioconjugated quantum dots

Present work demonstrates the utilization of surface modified polycarbonate (PC) membrane as solid phase and antibody conjugated CdSe/ZnS quantum dots (QDs) as fluorescent label for the sensitive and selective detection of Salmonella typhi (S. typhi) in water in a period of 2.5 h. PC membrane was surface modified with glycine and activated by EDC/NHS for immobilization of S. typhi specific IgG. Antibody immobilized porous PC membrane was incubated with bacteria contaminated water for immunocapturing of S. typhi. Antibody conjugated QDs were also prepared by using carbodiimide chemistry. Both modified PC membrane and quantum dots were characterized by using various modern analytical tools. It was estimated that 1.95 molecules of QDs were successfully bio-conjugated per unit of IgG. PC membrane with captured bacteria was incubated with prepared IgG conjugated QDs for the formation of sandwich complex. Analysis of the regions of interest (ROI) in fluorescent micrographs showed that newly developed method based on PC and fluorescent QDs has 100 times higher detection sensitivity (100 cells/mL) as compared with detection using conventional dye (FITC) based methods.     

Highly conducting gold nanoparticles-graphene nanohybrid films for ultrasensitive detection of carcinoembryonic antigen

A new label-free amperometric immunosensor was developed for detection of carcinoembryonic antigen (CEA) based on chitosan-ferrocene (CS-Fc) and nano-TiO₂ (CS-Fc + TiO₂) complex film and gold nanoparticles-graphene (Au-Gra) nanohybrid. CS-Fc + TiO₂ composite membrane was first modified on a bare glass carbon electrode. Then Au-Gra nanohybrid was formed on the CS-Fc + TiO₂ membrane by self-assembly strategy. Next, further immobilization of anti-CEA was constructed according to the strong interaction between Au-Gra and the amido groups of anti-CEA. Since Au-Gra nanohybrid films provided a congenial microenvironment for the immobilization of biomolecules, the surface coverage of antibody protein could be enhanced and the sensitivity of the immunosensor has been improved. The good electronic conductive characteristic might be attributed to the synergistic effect of graphene nanosheets and Au NPs. The modified process was characterized by scanning electron microscope (SEM) and cyclic voltammetry (CV). Under optimized conditions, the resulting biosensor displayed good amperometric response to CEA with linear range from 0.01 to 80 ng/mL and a detection limit of 3.4 pg/mL (signal/noise = 3). The results demonstrated that the immunosensor has advantages of high conduction, sensitivity, and long life time. This assay approach showed a great potential in clinical applications and detection of low level proteins.     

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW^® Malaria).     

玉米赤霉烯酮人工抗原的合成以及抗体的制备

制备了玉米赤霉烯酮人工抗原,并且制备抗血清,为进一步建立酶联免疫分析方法奠定基础.应用活泼酯法和混合酸酐法分别合成人工免疫原和人工检测原,进行动物免疫获得抗血清.结果表明,经紫外吸收、红外光谱、荧光光谱和酶联免疫方法验证人工抗原合成成功,玉米赤霉烯酮-牛血清白蛋白、玉米赤霉烯酮-卵清蛋白偶联比分别为12:1和8:1,抗...