Entwicklung von Oligonukleotidarrays für den Mikrobennachweis

Anordnungen von biomolekularen Sonden auf Festkörperoberflächen (Arrays, Chips) sind das Herzstück von Konzepten zum Aufbau von parallel arbeitenden miniaturisierten Analysesystemen, die die heute üblichen Verfahren beim chemisch-biologischen Screening und der molekularen Diagnostik ersetzen werden. Die Arrays werden nach der Art der immobilisierten Sonden bzw. nach Art der Herstellung der Sonden unterteilt. Die DNA-Sonden werden entweder in situ mittels photolithographischer Methoden unter Verwendung physikalischer Masken auf der Matrix synthetisiert oder durch verschiedene Verfahren aufgedruckt. Die Herstellung von gedruckten DNA Mikroarrays gliedert sich in die Schritte der Aktivierung bzw. Beschichtung der festen Chipmatrix, an die Biomoleküle über eine geeignete Kopplungschemie fixiert werden. Zur Herstellung von Oligonukleotidarrays für den Mikrobennachweis wurden Glasobjektträger mit Polymermaterialien beschichtet und mit unmodifizierten oder amino-modifizierten 18-50mer Oligonukleotiden bedruckt. Die Oligonukleotide wurden mit dem piezoelektrischen BioChip Arrayer von Packard in Volumina von 0,35-2 nl auf dem Chip aufgebracht. Die Spotmorphologie wurde in Abhängigkeit vom Spotvolumen, dem Druckpuffer und der Polymereigenschaften untersucht

Miniaturized Protein Microarray with Internal Calibration as Point-of-Care Device for Diagnosis of Neonatal Sepsis

Neonatal sepsis is still a leading cause of death among newborns. Therefore a protein-microarray for point-of-care testing that simultaneously quantifies the sepsis associated serum proteins IL-6, IL-8, IL-10, TNF alpha, S-100, PCT, E-Selectin, CRP and Neopterin has been developed. The chip works with only a 4 μL patient serum sample and hence minimizes excessive blood withdrawal from newborns. The 4 μL patient samples are diluted with 36 μL assay buffer and distributed to four slides for repetitive measurements. Streptavidin coated magnetic particles that act as distinct stirring detection components are added, not only to stir the sample, but also to detect antibody antigen binding events. We demonstrate that the test is complete within 2.5 h using a single step assay. S-100 conjugated to BSA is spotted in increasing concentrations to create an internal calibration. The presented low volume protein-chip fulfills the requirements of point-of-care testing for accurate and repeatable (CV < 14%) quantification of serum proteins for the diagnosis of neonatal sepsis

Challenges in Developing a Biochip for Intact Histamine Using Commercial Antibodies

This study describes the development and the challenges in the development of an on-chip immunoassay for histamine using commercially available antibodies. Histamine can be used as an indicator of food freshness and quality, but it is also a relevant marker in clinical diagnostics. Due to its low molecular weight, simple structure and thus low immunogenicity production of high specificity and affinity antibodies is difficult. From six commercial anti-histamine antibodies tested, only two bound the histamine free in the solution. A fluorescent on-chip immunoassay for histamine was established with a dynamic range of 8–111 µg/mL using polyclonal anti-histamine antibody H7403 from Sigma (Mendota Heights, MN, USA). The anti-histamine antibodies described and used in published literature are thoroughly reviewed and the quality of commercial antibodies and their traceability and quality issues are highlighted and extensively discussed

A Chip for Estrogen Receptor Action: Detection of Biomarkers Released by MCF-7 Cells through Estrogenic and Anti-Estrogenic Effects

The fluorescence-based multi-analyte chip platform for the analysis of estrogenic and anti-estrogenic substances is a new in vitro tool for the high throughput screening of environmental samples. In contrast to existing tools, the chip investigates the complex action of xenoestrogens in a human cell model by characterizing protein expression. It allows for the quantification of 10 proteins secreted by MCF-7 cells, representing various biological and pathological endpoints of endocrine action and distinguishing between estrogen- and anti-estrogen-dependent secretion of proteins. Distinct protein secretion patterns of the cancer cell line after exposure to known estrogen receptor agonists ß-estradiol, bisphenol A, genistein, and nonylphenol as well as antagonists fulvestrant and tamoxifen demonstrate the potential of the chip. Stimulation of cells with Interleukin-1ß shifts concentrations of low abundant biomarkers towards the working range of the chip. In the non-stimulated cell culture, Matrix Metalloproteinase 9 (MMP-9) and Vascular Endothelial Growth Factor (VEGF) show differences upon treatment with antagonists and agonists of the estrogen receptor. In stimulated MCF-7 cells challenged with receptor agonists secretion of Monocyte Chemoattractant Protein (MCP-1), Interleukin-6 (IL-6), Rantes, and Interleukin-8 (IL-8) significantly decreases. In parallel, the proliferating effect of endocrine-disrupting substances in MCF-7 cells is assessed in a proliferation assay based on resazurin. Using ethanol as a solvent for test substances increases the background of proliferation and secretion experiments, while using dimethyl sulfoxide (DMSO) does not show any adverse effects. The role of the selected biomarkers in different physiological processes such as cell development, reproduction, cancer, and metabolic syndrome makes the chip an excellent tool for either indicating endocrine-disrupting effects in food and environmental samples, or for screening the effect of xenoestrogens on a cellular and molecular level

Investigating Colorimetric Protein Array Assay Schemes for Detection of Recurrence of Bladder Cancer

A colorimetric microarray for the multiplexed detection of recurrence of bladder cancer including protein markers interleukin-8 (IL8), decorin (DCN), and vascular endothelial growth factor (VEGF) was established to enable easy and cheap read-out by a simple office scanner paving the way for quick therapy monitoring at doctors’ offices. The chip is based on the principle of a sandwich immunoassay and was optimized prior to multiplexing using IL8 as a model marker. Six different colorimetric assay formats were evaluated using a detection antibody (dAB) labeled with (I) gold (Au) nanoparticles (NPs), (II) carbon NPs, (III) oxidized carbon NPs, and a biotinylated dAB in combination with (IV) neutravidin–carbon, (V) streptavidin (strp)–gold, and (VI) strp–horseradish peroxidase (HRP). Assay Format (III) worked best for NP-based detection and showed a low background while the enzymatic approach, using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, led to the most intense signals with good reproducibility. Both assay formats showed consistent spot morphology as well as detection limits lower than 15 ng/L IL8 and were thus applied for the multiplexed detection of IL8, DCN, and VEGF in synthetic urine. Colorimetric detection in urine (1:3) yields reaction signals and measurement ranges well comparable with detection in the assay buffer, as well as excellent data reproducibility as indicated by the coefficient of variation (CV 5–9%)

Ammonia fluorosensors based on reversible lactonization of polymer-entrapped rhodamine dyes, and the effects of plasticizers

A new kind of optical sensor for monitoring dissolved ammonia uses rhodamines immobilized in thin membranes made from ethylcellulose, poly(vinyl chloride) or poly(vinyl acetate). The response to ammonia is the result of a change in the molecular structure of the rhodamine. On exposure to ammonia, the rhodamine is converted to a colorless and non-fluorescent lactone. As a result, fluorescence intensity is reduced. The polymer not only has a strong effect on the limits of detection, but also on the response time. Typical LODs are 0.1 μg ml−1 for ethylcellulose and poly(vinyl chloride), but as low as 10 μg ml−1 for poly(vinyl acetate). The last membranes provide highest sensitivity and lowest detection limits, but are not suitable for online monitoring of ammonia because they respond irreversibly. They may be applied, however, for “single shot” tests. Response times of the membranes are in the order of 2–5 min

Simulation and Performance Optimization of an Amperometric Histamine Detection System

One of the most widely known biogenic amines is histamine, which plays an important role in the human immune system. Some people suffer from allergic reactions after a histamine-rich diet; this is called histamine intolerance. The aim of this work is to develop a quick and reliable method for the detection and quantification of histamine in food, based on an electrochemical approach. In presence of biogenic amines, a reduction cascade induces a current at the working electrode. Prior to chronoamperometric measurements, Finite Elemente simulations were performed. The results are presented in this work