Biosensors2015, 5(2), 131-140; doi:10.3390/bios5020131 - published 26 March 2015 Show/Hide Abstract
Abstract: An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose “Q’-body”, which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q’-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q’-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.
Biosensors2015, 5(1), 118-130; doi:10.3390/bios5010118 - published 23 March 2015 Show/Hide Abstract
Abstract: Rapid biosensing requires fast mass transport of the analyte to the surface of the sensing element. To optimize analysis times, both mass transport in solution and the geometry and size of the sensing element need to be considered. Small dielectric spheres, tens of microns in diameter, can act as label-free biosensors using whispering gallery mode (WGM) resonances. WGM resonances are sensitive to the effective refractive index, which changes upon analyte binding to recognition sites on functionalized resonators. The spherical geometry and tens of microns diameter of these resonators provides an efficient target for sensing while their compact size enables detection in limited volumes. Here, we explore conditions leading to rapid analyte detection using WGM resonators as label-free sensors in 10 μL sample droplets. Droplet evaporation leads to potentially useful convective mixing, but also limits the time over which analysis can be completed. We show that active droplet mixing combined with initial binding rate measurements is required for accurate nanomolar protein quantification within the first minute following injection.
Biosensors2015, 5(1), 98-117; doi:10.3390/bios5010098 - published 23 March 2015 Show/Hide Abstract
Abstract: Label-free detection of rare cells in biological samples is an important and highly demanded task for clinical applications and various fields of research, such as detection of circulating tumor cells for cancer therapy and stem cells studies. Surface Plasmon Resonance (SPR) as a label-free method is a promising technology for detection of rare cells for diagnosis or research applications. Short detection depth of SPR (400 nm) provides a sensitive method with minimum interference of non-targets in the biological samples. In this work, we developed a novel microfluidic chip integrated with gold nanoslit SPR platform for highly efficient immunomagnetic capturing and detection of rare cells in human blood. Our method offers simple yet efficient detection of target cells with high purity. The approach for detection consists of two steps. Target cells are firs captured on functionalized magnetic nanoparticles (MNPs) with specific antibody I. The suspension containing the captured cells (MNPs-cells) is then introduced into a microfluidic chip integrated with a gold nanoslit film. MNPs-cells bind with the second specific antibody immobilized on the surface of the gold nanoslit and are therefore captured on the sensor active area. The cell binding on the gold nanoslit was monitored by the wavelength shift of the SPR spectrum generated by the gold nanoslits.
Biosensors2015, 5(1), 85-97; doi:10.3390/bios5010085 - published 19 March 2015 Show/Hide Abstract
Abstract: We demonstrate a simple force-based label-free strategy for the highly sensitive sensing of adenosine. An adenosine ssDNA aptamer was bound onto an atomic force microscopy (AFM) probe by covalent modification, and the molecular-interface adsorption force between the aptamer and a flat graphite surface was measured by single-molecule force spectroscopy (SMFS). In the presence of adenosine, the molecular recognition between adenosine and the aptamer resulted in the formation of a folded, hairpin-like DNA structure and hence caused a variation of the adsorption force at the graphite/water interface. The sensitive force response to molecular recognition provided an adenosine detection limit in the range of 0.1 to 1 nM. The addition of guanosine, cytidine, and uridine had no significant interference with the sensing of adenosine, indicating a strong selectivity of this sensor architecture. In addition, operational parameters that may affect the sensor, such as loading rate and solution ionic strength, were investigated.
Biosensors2015, 5(1), 69-84; doi:10.3390/bios5010069 - published 5 February 2015 Show/Hide Abstract
Abstract: Food defense requires the means to efficiently screen large volumes of food for microbial pathogens. Even rapid detection methods often require lengthy enrichment steps, making them impractical for this application. There is a great need for rapid, sensitive, specific, and inexpensive methods for extracting and concentrating microbial pathogens from food. In this study, an immuno-magnetic separation (IMS) methodology was developed for Escherichia coli O157:H7, using electrically active magnetic nanoparticles (EAMNPs). The analytical specificity of the IMS method was evaluated against Escherichia coli O55:H7 and Shigella boydii, and was improved over previous protocols by the addition of sodium chloride during the conjugation of antibodies onto MNPs. The analytical sensitivity of the IMS method was greatest when a high concentration of antibodies (1.0 mg/mL) was present during conjugation. EAMNP concentrations of 1.0 and 0.5 mg/mL provided optimal analytical sensitivity and analytical specificity. The entire IMS procedure requires only 35 min, and antibody-conjugated MNPs show no decline in performance up to 149 days after conjugation. This analytically sensitive and specific extraction protocol has excellent longevity and shows promise as an effective extraction for multiple electrochemical biosensor applications.
Biosensors2015, 5(1), 51-68; doi:10.3390/bios5010051 - published 5 February 2015 Show/Hide Abstract
Abstract: The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined, and primers based on the wzx (O-antigen flippase) and/or wzy (O-antigen polymerase) genes within the O-antigen gene clusters were designed and used in PCR assays to identify each serogroup. Specificity was tested with E. coli reference strains, field isolates belonging to the target serogroups, and non-E. coli bacteria. The PCR assays were highly specific for the respective serogroups; however, the PCR assay targeting the O62 wzx gene reacted positively with strains belonging to E. coli O68, which was determined by serotyping. Analysis of the O-antigen gene cluster sequences of serogroups O62 and O68 reference strains showed that they were 94% identical at the nucleotide level, although O62 contained an insertion sequence (IS) element located between the rmlA and rmlC genes within the O-antigen gene cluster. A PCR assay targeting the rmlA and rmlC genes flanking the IS element was used to differentiate O62 and O68 serogroups. The PCR assays developed in this study can be used for the detection and identification of E. coli O62/O68, O131, O140, O142, and O163 strains isolated from different sources.