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Vashist S.K.,Hsg Imit Institute For Mikro Und Informationstechnik | Vashist S.K.,Albert Ludwigs University of Freiburg | Schneider E.M.,University of Ulm | Lam E.,National Research Council Canada | And 2 more authors.
Scientific Reports | Year: 2014

An improved enzyme-linked immunosorbent (ELISA) assay using one-step antibody immobilization has been developed for the detection of human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The anti-HFA formed a stable complex with 3-aminopropyltriethoxysilane (APTES) by ionic and hydrophobic interactions. The complex adsorbed on microtiter plates exhibited a detection range of 4.9 pg mL-1 to 20 ng mL-1 HFA, with a limit of detection of 7 pg mL-1. Furthermore, an analytical sensitivity of 10 pg mL-1 was achieved, representing a 51-fold increase in sensitivity over the commercial sandwich ELISA kit. The results obtained for HFA spiked in diluted human whole blood and plasma showed the same precision as the commercial kit. When stored at 4°C in 0.1 M phosphate-buffered saline (PBS, pH 7.4), the anti-HFA bound microtiter plates displayed no significant decrease in their functional activity after two months. The new ELISA procedure was extended for the detection of C-reactive protein, human albumin and human lipocalin-2 with excellent analytical performance.


Vashist S.K.,Hsg Imit Institute For Mikro Und Informationstechnik | Vashist S.K.,Albert Ludwigs University of Freiburg | Luong J.H.T.,University College Cork
Carbon | Year: 2015

The last decade has witnessed tremendous advances in the use of graphene and graphenebased nanocomposites for the fabrication of electrochemical (EC) sensors and biosensors with improved analytical performance. With unique and highly desirable morphology, chemical/thermal stability and EC properties, the graphene-based materials are paving way to the implementation of mediatorless EC detection schemes with direct electron transfer. This approach enables the development of highly performed biosensors with respect to detection sensitivity, precision, specificity, and stability. This review provides a comprehensive overview of the field apart from providing intensive information of the fabrication, properties, characterization and EC applications of graphene and its nanocomposites. Two key challenges, the lack of international regulatory guidelines for nanotoxicity analysis and potential mass production of analytical devices, will also be discussed along with the trends in nanobiotechnology and the requirements in healthcare and industrial applications. © 2014 Elsevier Ltd. All rights reserved.


Escadafal C.,Robert Koch Institute | Faye O.,Institute Pasteur Of Dakar | Sall A.A.,Institute Pasteur Of Dakar | Weidmann M.,University of Gottingen | And 9 more authors.
PLoS Neglected Tropical Diseases | Year: 2014

Background:Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV), is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control.Methodology:The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA) assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay) to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal.Conclusion/Significance:The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction) and rapid processing time (<20 min). Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for YFV detection in low-resource settings. © 2014 Escadafal et al.


Vashist S.K.,Hsg Imit Institute For Mikro Und Informationstechnik | Mudanyali O.,University of California at Los Angeles | Schneider E.M.,University of Ulm | Zengerle R.,Hsg Imit Institute For Mikro Und Informationstechnik | And 2 more authors.
Analytical and Bioanalytical Chemistry | Year: 2014

During the last decade, there has been a rapidly growing trend toward the use of cellphone-based devices (CBDs) in bioanalytical sciences. For example, they have been used for digital microscopy, cytometry, read-out of immunoassays and lateral flow tests, electrochemical and surface plasmon resonance based bio-sensing, colorimetric detection and healthcare monitoring, among others. Cellphone can be considered as one of the most prospective devices for the development of next-generation point-of-care (POC) diagnostics platforms, enabling mobile healthcare delivery and personalized medicine. With more than 6.5 billion cellphone subscribers worldwide and approximately 1.6 billion new devices being sold each year, cellphone technology is also creating new business and research opportunities. Many cellphone-based devices, such as those targeted for diabetic management, weight management, monitoring of blood pressure and pulse rate, have already become commercially-available in recent years. In addition to such monitoring platforms, several other CBDs are also being introduced, targeting e.g., microscopic imaging and sensing applications for medical diagnostics using novel computational algorithms and components already embedded on cellphones. This report aims to review these recent developments in CBDs for bioanalytical sciences along with some of the challenges involved and the future opportunities. [Figure not available: see fulltext.] © 2013 Springer-Verlag Berlin Heidelberg.


Vashist S.K.,Dublin City University | Vashist S.K.,Hsg Imit Institute For Mikro Und Informationstechnik
Analytical Biochemistry | Year: 2014

We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human lipocalin-2 (LCN2) in plasma, serum, and whole blood. It has the dynamic range, linear range, limit of detection, and analytical sensitivity of 0.6 to 5120, 80 to 2560, 0.7, and 1 pg/ml, respectively. It is the most sensitive assay for the detection of LCN2, which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit. The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3-aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on MTP and GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody by its carboxyl groups using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as the heterobifunctional crosslinker. The anti-LCN2 antibody-bound MTPs were highly stable given that they did not show any significant decrease in their functional activity when stored at 4 C in 0.1 M phosphate-buffered saline (PBS) for 8 weeks. The developed immunoassay correlated well with the conventional ELISA, thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings. © 2013 Elsevier Inc. All rights reserved.

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