Micro/Nanoscale Parallel Patterning of Functional Biomolecules, Organic Fluorophores and Colloidal Nanocrystals

We describe the design and optimization of a reliable strategy that combines self-assembly and lithographic techniques, leading to very precise micro-/nanopositioning of biomolecules for the realization of micro- and nanoarrays of functional DNA and antibodies. Moreover, based on the covalent immobilization of stable and versatile SAMs of programmable chemical reactivity, this approach constitutes a general platform for the parallel site-specific deposition of a wide range of molecules such as organic fluorophores and water-soluble colloidal nanocrystals

The Diagnostic Sensitivity of Dengue Rapid Test Assays Is Significantly Enhanced by Using a Combined Antigen and Antibody Testing Approach

Dengue is a serious public health concern with around 3 billion people at risk of infection. Severe forms of the infection can be fatal and with no licensed vaccine or effective therapeutic currently available, early detection is important to assist with the clinical management of symptoms. Isolation of the virus and the detection of viral RNA using RT-PCR are commonly used methods for early diagnosis but are time-consuming, expensive and require skilled operation. Rapid immunochromatographic tests (ICT) are relatively simple, inexpensive and easy to perform at or near the point of care. Here, we report on the clinical performance of a new rapid ICT for the non-structural protein 1 (NS1) of dengue virus, a marker of acute infection. At two clinical study sites, NS1 was detected in 60–70% of laboratory-confirmed dengue cases and specificity of the test was >95%. We have also shown that a combined testing approach for both circulating NS1 antigen and antibody responses to the glycoprotein E of the virus can significantly improve diagnostic sensitivity compared to the detection of NS1 alone. Importantly, the combined antigen and antibody testing approach also provides an expanded window of detection from as early as day 1 post-onset of illness

Development of electrochemical biosensors with various types of zeolites

In the work, different types of zeolites were used for the development of enzyme-based electrochemical biosensors. Zeolites were added to the biorecognition elements of the biosensors and served as additional components of the biomembranes or adsorbents for enzymes. Three types of biosensors (conductometric, amperometric and potentiometric) were studied. The developed biosensors were compared with the similar biosensors without zeolites. The biosensors contained the following enzymes: urease, glucose oxidase, glutamate oxidase, and acetylcholinesterase and were intended for the detection of urea, glucose, glutamate, and acetylcholine, respectively. Construction of the biosensors using the adsorption of enzymes on zeolites has several advantages: simplicity, good reproducibility, quickness, absence of toxic compounds. These benefits are particularly important for the standardization and further mass production of the biosensors. Furthermore, a biosensor for the sucrose determination contained a three-enzyme system (invertase/mutatorase/glucose oxidase), immobilized by a combination of adsorption on silicalite and cross-linking via glutaraldehyde; such combined immobilization demonstrated better results as compared with adsorption or cross-linking separately. The analysis of urea and sucrose concentrations in the real samples was carried out. The results, obtained with biosensors, had high correlation with the results of traditional analytical methods, thus the developed biosensors are promising for practical applications

Gold Nanoparticle/Polymer/Enzyme Nanocomposite for the Development of Adenosine Triphosphate Biosensor

International audienceDevelopment of enzyme-containing nanocomposites provides an excellent opportunity for development of sensitive and effective analytical devices – biosensors. In our work we used nanocomposites that contain two enzymes, polymers and gold nanoparticles (about 20 nm). Such nanostructure was expected to increase electron transfer in the bioselective element of biosensor and to improve enzyme stability during the immobilization process. In the present work, an amperometric biosensor based on a bienzymatic system (glucose oxidase/hexokinase) was developed. The biosensor was sensitive to adenosine-5'-triphosphate (ATP). The enzymes were immobilized onto the surface of a platinum disc electrode which was used as amperometric transducer. Three different methods of immobilization were investigated: cross-linking of the enzymes with bovine serum albumin, entrapment in a photo-crosslinkable polyvinyl alcohol (PVA) matrix, and entrapment of the enzymes in a PVA / polyethylenimine matrix. All the methods were examined with and without addition of gold nanoparticles (GNPs) to the reacting mixture for enzymatic membrane creation. GNPs were added to decrease insulating properties of polymer/enzyme films and improve electron transfer between enzymes and electrode. ATP detection was achieved in all cases, with good reproducibility but variable time of response, the highest sensitivity being achieved by co-immobilizing glucose oxidase/hexokinase into the photo-crosslinked PVA/GNPs polymer matrix.The authors gratefully acknowledge the financial support of this study by Project European IRSES-NANODEVICE and by NASU in the frame of Scientific and Technical Program “Sensor devices for medical-ecological and industrial purposes: metrology and trial performance

Application of PCR in serum samples for diagnosis of paracoccidioidomycosis in the southern Bahia-Brazil.

Paracoccidioidomycosis (PCM) cannot always be diagnosed by conventional means such as direct examination of histopathology or clinical samples, and serological methods, used as an alternative, still have many cases of cross-reactivity. In this scenario, molecular techniques seem to arise as a rapid approach, specific and direct that could be used in the diagnosis of this mycosis. In this study we analyzed 76 serum samples from patients in southern Bahia suspected of having paracoccidioidomycosis using a conventional PCR with primers for the ITS1 ribosomal DNA of P. brasiliensis. Of these 76 patients, 5 were positive for PCM by double immunodiffusion and/or direct examination and histopathology. To test specificity of PCR, we used human DNA and three isolates of P. lutzii (1578, 01 and ED01). Additionally, we analyzed by serial dilutions of DNA the limit of detection of the assay. The test of PCR proved specific, as only a 144 bp fragment of the three isolates of P. lutzii and no human DNA was amplified. Detection limit was 1.1 pg/µL of DNA. Despite the high detection limit and specificity of PCR none of the 76 serum samples were found positive by PCR, but a biopsy specimen obtained from one of the patients with PCM was positive. These results, albeit limited, show that PCR is not effective in detecting DNA of P. brasiliensis or P. lutzii in serum, but could perhaps be used with other types of clinical samples, especially in those instances in which conventional methods fail