In contrast to mammals, early B cell differentiation and diversification of the antibody repertoire in chickens do not take place in the bone marrow but in a specialized gut associated lymphoid ...tissue (GALT), the bursa of Fabricius. During embryonic development, B cell precursors migrate to the bursa anlage, where they proliferate and diversify their B cell receptor repertoire. Around hatch these diversified B cells start to emigrate from the bursa of Fabricius and populate peripheral lymphoid organs, but very little is known how the migratory processes are regulated. As CXCL12 (syn. SDF-1) and CXCR4 were shown to be essential for the control of B cell migration during the development of lymphoid tissues in mammals, we analyzed expression and function of this chemokine/chemokine-receptor pair in the chicken bursa. We found a strong variation of mRNA abundance of CXCL12 and CXCR4 in different stages of bursa development, with high abundance of CXCL12 mRNA in the bursa anlage at embryonic day 10 (ED10).
hybridization demonstrated disseminated CXCL12 expression in the early bursa anlage, which condensed in the developing follicles and was mainly restricted to the follicle cortex post-hatch. Flow cytometric analysis detected CXCR4 protein already on early B cell stages, increasing during bursal development. Post-hatch, a subpopulation with the hallmarks of emigrating B cells became detectable, which had lower CXCR4 expression, suggesting that downregulation of CXCR4 is necessary to leave the CXCL12-high bursal environment.
blockade of CXCR4 using AMD3100 at the time of B cell precursor immigration strongly inhibited follicle development, demonstrating that CXCL12 attracts pre-bursal B cells into the bursal anlage. Altogether, we show that CXCL12 and its receptor CXCR4 are important for both populating the bursa with B cells and emigration of mature B cells into the periphery post hatch, and that CXCR4 function in primary B cell organs is conserved between mammals and birds.
MiRNAs are non-coding RNA molecules that control biological functions by reducing the translation of target proteins when binding to the mRNA. Alterations of the miRNA expression profile affect the ...cell metabolism, which can lead to distinctive disease patterns thus suggesting miRNA as an interesting biomarker. Here we present a SPR biosensor that utilizes disposable, injection-molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one-dimensional spot arrays to detect miRNA-93. Using a RNA-DNA-hybrid antibody for signal enhancement we could reach a limit of detection of 10 pmol/l.
•Novel platform based on 1D photonic crystal biochips combining label-free and fluorescence detection.•VEGF cancer biomarker detection by means of specific antibodies on chip.•Detection of 2.5ng/mL ...VEGF in phosphate buffer saline demonstrated, with an estimated limit of detection (LoD) of 0.65ng/mL.•Detection of 3.5ng/mL VEGF in human plasma demonstrated.•The assay requires a short time (30min) and the disposable plastic chips are ready for mass production.
We report on the detection of an angiogenic molecule Vascular Endothelial Growth Factor (VEGF) in different biological matrices by means of a new integrated biosensing platform exploiting the properties of Bloch surface waves. The new platform takes advantage of a tandem configuration, in which both label-free and enhanced fluorescence detection are implemented. Specifically designed one dimensional photonic crystals were deposited directly on disposable and low cost plastic biochips. A direct sandwich immunoassay was used to detect VEGF in buffer, cell culture supernatant and human plasma at low concentration (ng/mL). The platform enabled the detection of VEGF in all three matrices with high resolution, fast turnaround time (30min) and in close agreement with the results of enzyme linked immunosorbent assays.
We describe the design and fabrication of biochips based on 1-D photonic crystals supporting Bloch surface waves for label-free optical biosensing. The optical properties of Bloch surface waves are ...studied in relation to the geometry of the photonic crystals and on the properties of the dielectric materials used for the fabrication. The planar stacks of the biochips are composed of silica, tantala, and titania that were deposited using plasma-ion-assisted evaporation under high-vacuum conditions. The biochip surfaces were functionalized by silanization, and appropriate fluidic cells were designed to operate in an automated platform. An angularly resolved ellipsometric optical sensing apparatus was assembled to carry out the sensing studies. The angular operation is obtained by a focused laser beam at a fixed wavelength and detection of the angular reflectance spectrum by means of an array detector. The results of the experimental characterization of the physical properties of the fabricated biochips show that their characteristics, in terms of sensitivity and figure of merit, match those expected from the numerical simulations. Practical application of the sensor was demonstrated by detecting a specific glycoprotein, Angio-poietin 2, that is involved in angiogenesis and inflammation processes. The protocol used for the label-free detection of Angiopoietin 2 is described, and the results of an exemplary assay, carried out at a relatively high concentration of 1 μg/ml, are given and confirm that an efficient detection can be achieved. The limit of detection of the biochips for Angiopoietin 2, based on the protocol used, is 1.5 pg/mm 2 in buffer solution. The efficiency of the label-free assay is confirmed by independent measurements carried out by means of confocal fluorescence microscopy.
In this work, an approach for SPR spectroscopy using the liSPR system is examined that combines signal amplification by PCR and magnetic nanoparticles in one injection step. Therefore, the synthesis ...of PCR products was performed on the beads similar to a solid‐phase PCR, termed PCR‐on‐a‐bead. The functionality of this PCR was proven using an enzymatic assay. For validation the detection of oligonucleotides by SPR, an asymmetric PCR product was investigated. A signal increase upon binding of the PCR product to the specific probes was observed. In addition, surface regeneration of the chip was examined and reuse for at least two times ascertained. Amplification of the SPR signal by magnetic beads was verified but no signal was detected for PCR products immobilized on particles prior to injection.
MiRNAs are endogenous noncoding RNA molecules. They play important gene‐regulatory roles by binding to the mRNA of target genes thereby leading to either transcript degradation or translational ...repression. In virtually all diseases, distinct alterations of miRNA expression profiles have been found thus suggesting miRNAs as interesting biomarkers. Here, we present an SPR biosensor that utilizes disposable, injection‐molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one‐dimensional spot arrays to detect miRNA‐93. To increase the sensitivity of the biosensor we used two different amplification strategies. By adding an RNA‐DNA‐hybrid antibody for primary signal amplification, a limit of detection of 10 pmol/L was achieved. Based on that method we demonstrate the detection of miRNA‐93 in total RNA lysate from HEK‐293 cells. Utilizing an enzymatic signal amplification with Poly(A) polymerase, the sensitivity could be increased even further leading to a limit of detection of 1 fmol/L.
The development of a surface plasmon resonance (SPR) spectrometer comprising angular‐resolved analysis for quasi‐monochromatic illumination is reported. The optical system utilizes disposable, ...injection‐molded chips combined with a lateral imaging optical system for parallel analysis of one‐dimensional spot arrays. Further parallelization is achieved by introducing a segmented light source. This source sequentially illuminates three neighbored one‐dimensional arrays in order to keep angular‐resolved analysis without introducing any mechanically moving parts. This system is applied to detect genetic variations among different DNA samples obtained from polymerase chain reaction (PCR). For this purpose, 135 spots on the chip surface have been prepared by spotting and analyzed separately.
Quantitative detection of angiogenic biomarkers provides a powerful tool to diagnose cancers in early stages and to follow its progression during therapy. Conventional tests require trained ...personnel, dedicated laboratory equipment and are generally time-consuming. Herein, we propose our developed biosensing platform as a useful tool for a rapid determination of Angiopoietin-2 biomarker directly from patient plasma within 30 minutes, without any sample preparation or dilution. Bloch surface waves supported by one dimensional photonic crystal are exploited to enhance and redirect the fluorescence arising from a sandwich immunoassay that involves Angiopoietin-2. The sensing units consist of disposable and low-cost plastic biochips coated with the photonic crystal. The biosensing platform is demonstrated to detect Angiopoietin-2 in plasma samples at the clinically relevant concentration of 6 ng/mL, with an estimated limit of detection of approximately 1 ng/mL. This is the first Bloch surface wave based assay capable of detecting relevant concentrations of an angiogenic factor in plasma samples. The results obtained by the developed biosensing platform are in close agreement with enzyme-linked immunosorbent assays, demonstrating a good accuracy, and their repeatability showed acceptable relative variations.
► A new SPR platform ‘Phytochip’ was developed and established. ► It provides an improved detection assay to distinguish virus-infected plants from non-infected plants. ► It has a short detection ...time and can be used in a high throughput system to analyze efficiently many samples. ► Phytochip is suitable for practical application in plant breeding and virus control.
The surface plasmon resonance (SPR) based ‘Phytochip’ was developed to distinguish virus-infected plants from non-infected plants. The system detects DNA–RNA hybridization to show the presence of phytopathogenic viruses such as the RNA virus barley stripe mosaic virus (BSMV) in wheat leaves. To achieve this BSMV and wheat specific oligonucleotides, and a negative control yeast oligonucleotide, were immobilized on a SPR gold surface chip. After optimization of the hybridization parameters with purified wheat samples, wheat infected with BSMV resulted in detectable signals with both the BSMV and the wheat probes. In contrast, a hybridization reaction was not be detected with the negative probe. The method is fast and sensitive with a detection time of 3000s (50min), a detection limit of 14.7pgμl−1 BSMV RNA and a measuring range of 14.7–84pgμl−1 BSMV RNA (1.323–7.56ng BSMV RNA per 90μl sample). These characteristics, combined with the high throughput design, make it suitable for application in plant breeding and virus control.
Aptamers are synthetic single‐stranded oligonucleotides which bind specifically to their target. They offer several advantages over antibodies. For example, aptamers can be produced under ...unphysiological conditions against almost any target, including toxic or pathological substances. They are also quicker and cheaper produced than antibodies, and are easy to modify without loss of activity. Furthermore, they exhibit high stability under a width range of conditions. Consequently, they make excellent receptors for the use in biosensors. This article describes the evaluation of a novel aptasensor based on the Surface Plasmon Resonance (SPR)‐system developed by the Fraunhofer Institute for Applied Optics and Precision Engineering IOF (Jena, Germany) using a thrombin–aptamer interaction as a model system. The biotin‐tagged aptamer was attached to the sensor's gold surface by means of its interaction with streptavidin. Thrombin solutions of different concentrations were pumped over this surface, and the interaction was measured under buffer flow. The binding signals for the thrombin–aptamer interaction were compared to those arising from a control random‐oligonucleotide of the same size and bearing the same modifications. Using this approach, we were able to obtain reproducible, significant and stable signals with a limit of detection of about 26 nmol/L.
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