Exploring Simple Particle-Based Signal Amplification Strategies in a Heterogeneous Sandwich Immunoassay with Optical Detection.

Heterogeneous sandwich immunoassays are widely used for biomarker detection in bioanalysis and medical diagnostics. The high analyte sensitivity of the current "gold standard" enzyme-linked immunosorbent assay (ELISA) originates from the signal-generating enzymatic amplification step, yielding a high number of optically detectable reporter molecules. For future point-of-care testing (POCT) and point-of-need applications, there is an increasing interest in more simple detection strategies that circumvent time-consuming and temperature-dependent enzymatic reactions. A common concept to aim for detection limits comparable to those of enzymatic amplification reactions is the usage of polymer nanoparticles (NP) stained with a large number of chromophores. We explored different simple NP-based signal amplification strategies for heterogeneous sandwich immunoassays that rely on an extraction-triggered release step of different types of optically detectable reporters. Therefore, streptavidin-functionalized polystyrene particles (PSP) are utilized as carriers for (i) the fluorescent dye coumarin 153 (C153) and (ii) hemin (hem) molecules catalyzing the luminol reaction enabling chemiluminescence (CL) detection. Additionally, (iii) NP labeling with hemin-based microperoxidase MP11 was assessed. For each amplification approach, the PSP was first systematically optimized regarding size, loading concentration, and surface chemistry. Then, for an immunoassay for the inflammation marker C-reactive protein (CRP), the analyte sensitivity achievable with optimized PSP systems was compared with the established ELISA concept for photometric and CL detection. Careful optimization led to a limit of detection (LOD) of 0.1 ng/mL for MP11-labeled PSP and CL detection, performing similarly well to a photometric ELISA (0.13 ng/mL), which demonstrates the huge potential of our novel assay concept.

Characterization of bitter peptides in casein hydrolysates using comprehensive two-dimensional liquid chromatography.

Casein hydrolysates are important additives to foods for elderly and sports nutrition. However, due to the enzymatic generation of so-called bitter peptides, their application is limited. Therefore, the procedure needs to be optimized in order to restrict their occurrence. For this, extensive sensory evaluations are necessary. By combining two separation techniques using comprehensive two-dimensional liquid chromatography, we present a novel method for estimating the bitter taste of hydrolysate samples on the basis of their elution pattern. Using a size exclusion column in the first and a reversed phase column in the second dimension allows for a detailed sample evaluation regarding peptide size and relative hydrophobicity. The results obtained for different casein hydrolysates were correlated with the sensory evaluation. We found that hydrolysates with increasing bitterness contain a higher amount of peptides of high hydrophobicity and a molecular weight less than 6.5 kDa.

Effect of glucose depletion during the synthesis of galactooligosaccharides using a trienzymatic system.

In this study the effect of glucose depletion using glucose oxidase and catalase, simultaneously to the synthesis of prebiotic galactooligosaccharides (GOS) by ß-galactosidase was studied. Considering total GOS yield, a strong dependency on the source of ß-galactosidase was found. Using an Aspergillus oryzae lactase, a small increase in GOS yield (from 50.0 ± 1.3 g/L to 54.1 ± 1.9 g/L) was noted. Due to the decreased rate of GOS disaccharide formation by the Kluyveromyces lactis enzyme, the total GOS yield was diminished (from 47.4 ± 0.1 g/L to 30.5 ± 1.7 g/L). However, for both enzymes, the synthesis of GOS tri- and higher oligosaccharides increased. Additionally, the total sugar content, and thus caloric value, of the final product was reduced, also resulting in a more purified GOS mixture.

Combination of two ß-galactosidases during the synthesis of galactooligosaccharides may enhance yield and structural diversity.

In this study, the combination of two ß-galactosidases to synthesize prebiotic galactooligosaccharides (GOS) was evaluated in terms of total GOS yield as well as GOS structures (chain length). Two different combinations of either Aspergillus oryzae and Cryptococcus laurentii or Aspergillus oryzae and Kluyveromyces lactis were tested to examine the influence of enzyme origin. Neither consecutive nor simultaneous synthesis with A. oryzae and C. laurentii led to an increased GOS yield. However, with the latter, synthesis of higher GOS (≥3 monomer units) was enhanced from 38.5% to 40% with special emphasis of tetra- and pentasaccharides, which increased from 6.7% to 12.8% and from 0.4% to 3.3%, respectively. Additionally, due to the different preferences of the two ß-galactosidases in terms of types of glycosidic linkages, the structural diversity of the final GOS product could be increased. Using K. lactis following the synthesis with A. oryzae increased the yield of total GOS from 24.6% to 33.1%, which was mainly due to the formation of GOS disaccharides. On the other hand, applying A. oryzae as the second enzyme led to a degradation of di- and trisaccharides, and thus total GOS yield was diminished, although the yield of tetrasaccharides could be enhanced. In conclusion, with both studied enzyme combinations it was possible to increase the percentage of higher GOS and reduce the residual lactose content of the final mixture, which is beneficial for subsequent purification processes. Thus, using more than one ß-galactosidase during the synthesis of GOS represents an interesting research area, which should be explored in more detail in the future.

CERA Attenuates Kidney Fibrogenesis in the db/db Mouse by Influencing the Renal Myofibroblast Generation.

Tubulointerstitial fibrosis (TIF) is a pivotal pathophysiological process in patients with diabetic nephropathy (DN). Multiple profibrotic factors and cell types, including transforming growth factor beta 1 (TGF-ß1) and interstitial myofibroblasts, respectively, are responsible for the accumulation of extracellular matrix in the kidney. Matrix-producing myofibroblasts can originate from different sources and different mechanisms are involved in the activation process of the myofibroblasts in the fibrotic kidney. In this study, 16-week-old db/db mice, a model for type 2 DN, were treated for two weeks with continuous erythropoietin receptor activator (CERA), a synthetic erythropoietin variant with possible non-hematopoietic, tissue-protective effects. Non-diabetic and diabetic mice treated with placebo were used as controls. The effects of CERA on tubulointerstitial fibrosis (TIF) as well as on the generation of the matrix-producing myofibroblasts were evaluated by morphological, immunohistochemical, and molecular biological methods. The placebo-treated diabetic mice showed significant signs of beginning renal TIF (shown by picrosirius red staining; increased connective tissue growth factor (CTGF), fibronectin and collagen I deposition; upregulated KIM1 expression) together with an increased number of interstitial myofibroblasts (shown by different mesenchymal markers), while kidneys from diabetic mice treated with CERA revealed less TIF and fewer myofibroblasts. The mechanisms, in which CERA acts as an anti-fibrotic agent/drug, seem to be multifaceted: first, CERA inhibits the generation of matrix-producing myofibroblasts and second, CERA increases the ability for tissue repair. Many of these CERA effects can be explained by the finding that CERA inhibits the renal expression of the cytokine TGF-ß1.

Synthesis of Galactooligosaccharides in Milk and Whey: A Review.

Galactooligosaccharides (GOS) are synthesized by the enzyme ß-galactosidase during the hydrolysis of lactose. In this so-called transgalactosylation reaction the galactosyl moiety is transferred to another sugar molecule instead of water resulting in oligosaccharides of different chain lengths and glycosidic linkages. Because their structures are similar to oligosaccharides present in human breast milk, they act as prebiotics, which has been shown for infants and adults to be alike. While so far most of the research to maximize GOS yield has been carried out using buffered lactose solution as a starting material, more and more work is now conducted with dairy by-products such as whey and whey permeate, or even milk, for direct GOS synthesis in order to develop new GOS-enriched dairy products. This review aims to summarize the results obtained with various dairy liquids, and it rates their suitabilities to act as raw material for GOS production. Most of the studies using whey or milk have been carried out with enzymes from Aspergillus oryzae, Kluyveromyces lactis, Bacillus circulans, Streptococcus thermophilus, and several Lactobacillus species. As the initial lactose concentration (ILC) is known to be a crucial factor for high GOS yield, most of the research has been done with concentrated or supplemented milk and whey. However, a clear dependency on ILC could only be observed for the A. oryzae lactase, indicating a strong influence of milk components like minerals and proteins on the transfer activities of most enzymes.

Aptamer-Based Trapping: Enrichment of Bacillus cereus Spores for Real-Time PCR Detection.

Aptamer-based trapping techniques are in general suitable to replace common antibody-based enrichment approaches. A time-consuming isolation or clean-up is often necessary during sample preparation, e.g. for the detection of spores. For the development of bioanalytical routine approaches, aptamers with a high affinity to B. cereus spores were applied for the establishment and validation of an aptamer-based trapping technique in milk with fat contents between 0.3 and 3.5%. Thereby, enrichment factors of up to sixfold were achieved. The combination of an aptamer-based enrichment by magnetic separation and the subsequent specific real-time PCR detection represents a reliable and rapid detection system.

Aptamers: Universal capture units for lateral flow applications.

The present work demonstrates the implementation of aptamers as capture molecules for a wide range of target classes in lateral flow assay applications. The targets were chosen in order to cover a wide range of target classes (small sized - metabolite, medium sized - protein, and large sized - whole cell/spore). For each target class one target molecule was selected as representative and appropriate aptamers were used for lateral flow assay development. The work points out that the implementation of aptamers as capture molecules in a universal lateral flow test platform was successful independent form target size. Furthermore, the limit of detection for p-aminohippuric acid in urine (200 ppm), lysozyme in white wine (20 ppm), and Alicyclobacillus spores in buffered orange juice (>8 CFU/mL) were determined using aptamers as capture molecules. The whole approach is considered as a proof of concept, regarding the ability of aptamers as an alternative to antibodies (in conjunction with directive 2010/63/EU on the protection of animals used for scientific purposes) in lateral flow applications.

Development of a Multiplex Real-Time PCR for Determination of Apricot in Marzipan Using the Plexor System.

Marzipan is a confectionary which is mostly offered in form of filled chocolate, pralines, or pure. According to the German guidelines for oil seeds only almonds, sugar and water are admitted ingredients of marzipan. A product very similar in taste is persipan which is used in the confectionary industry because of its stronger flavor. For persipan production almonds are replaced by debittered apricot or peach kernels. To guarantee high quality products for consumers, German raw paste producers have agreed a limit of apricot kernels in marzipan raw paste of 0.5%. Different DNA-based methods for quantitation of persipan contaminations in marzipan are already published. To increase the detection specificity compared to published intercalation dye-based assays, the present work demonstrate the utilization of a multiplex real-time PCR based on the Plexor technology. Thus, the present work enables the detection of at least 0.1% apricot DNA in almond DNA or less. By analyzing DNA mixtures, the theoretical limit of quantification of the duplex PCR for the quantitation of persipan raw paste DNA in marzipan raw paste DNA was determined as 0.05%.

Automated Enrichment of Sulfanilamide in Milk Matrices by Utilization of Aptamer-Linked Magnetic Particles.

The present work demonstrates the first automated enrichment approach for antibiotics in milk using specific DNA aptamers. First, aptamers toward the antibiotic sulfanilamide were selected and characterized regarding their dissociation constants and specificity toward relevant antibiotics via fluorescence assay and LC-MS/MS detection. The performed enrichment was automated using the KingFisherDuo and compared to a manual approach. Verifying the functionality, trapping was realized in different milk matrices: (i) 0.3% fat milk, (ii) 1.5% fat milk, (iii) 3.5% fat milk, and (iv) 0.3% fat cocoa milk drink. Enrichment factors up to 8-fold could be achieved. Furthermore, it could be shown that novel implementation of a magnetic separator increases the reproducibility and reduces the hands-on time from approximately half a day to 30 min.

Aptamer-based trapping of phytosphingosine in urine samples.

Usually, small molecules like single metabolites used in clinical diagnostic can be quantified by instrumental approaches like LC-MS or bioanalytical techniques using antibodies or aptamers as selective receptors. The present work comprises the generation of aptamers with an affinity towards the medically relevant metabolite phytosphingosine via the previously reported just in time-Selection approach (Hünniger et al., 2014). The whole approach could be seen as a proof of concept to extend the existing just in time-Selection protocol for selection towards small molecules with dissociation constants in the low nanomolar range. Moreover it is conceivable that the shown methods could be quickly adapted to further scopes. Aptamers could be applied for clean-up or concentration processes prior to further analysis. As an example, we used the selected aptamers towards phytosphingosine bound to magnetic particles for affinity enrichment in both selection buffer and urine samples. As an outcome, enrichment factors of up to 9-fold (selection buffer)/4-fold (urine samples) were achieved by this approach.

Food Sensing: Aptamer-Based Trapping of Bacillus cereus Spores with Specific Detection via Real Time PCR in Milk.

Aerobic spores pose serious problems for both food product manufacturers and consumers. Milk is particularly at risk and thus an important issue of preventive consumer protection and quality assurance. The spore-former Bacillus cereus is a food poisoning Gram-positive pathogen which mainly produces two different types of toxins, the diarrhea inducing and the emetic toxins. Reliable and rapid analytical assays for the detection of B. cereus spores are required, which could be achieved by combining in vitro generated aptamers with highly specific molecular biological techniques. For the development of routine bioanalytical approaches, already existing aptamers with high affinity to B. cereus spores have been characterized by surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in terms of their dissociation constants and selectivity. Dissociation constants in the low nanomolar range (from 5.2 to 52.4 nM) were determined. Subsequently, the characterized aptamers were utilized for the establishment and validation of an aptamer-based trapping technique in both milk simulating buffer and milk with fat contents between 0.3 and 3.5%. Thereby, enrichment factors of up to 6-fold could be achieved. It could be observed that trapping protocol and characterized aptamers were fully adaptable to the application in milk. Due to the fact that aptamer selectivity is limited, a highly specific real time PCR assay was utilized following trapping to gain a higher degree of selectivity.

Food sensing: selection and characterization of DNA aptamers to Alicyclobacillus spores for trapping and detection from orange juice.

The quality of the beverage industry's products has to be constantly monitored to fulfill consumers' high expectations. The thermo-acidophilic Gram-positive Alicyclobacillus spp. are not pathogenic, but their heat-resistant endospores can survive juice-processing conditions and have become a major economic concern for the fruit juice industry. Current detection methods rely on cultivation, isolation, and organism identification, which can take up to a week, resulting in economic loss. This work presents the selection and identification of DNA aptamers targeting Alicyclobacillus spores by spore-SELEX (systematic evolution of ligands by exponential enrichment) in orange-juice-simulating buffer. The selection process was verified by various techniques, including flow cytometric binding assays, radioactive binding assays, and agarose gel electrophoresis. The subsequent aptamer characterization included the determination of dissociations constants and selectivity by different techniques, such as surface plasmon resonance spectroscopy and fluorescence microscopy. In summary, 10 different aptamers with an affinity to Alicyclobacillus spp. have been developed, analyzed, and characterized in terms of affinity and specificity.

Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii.

BACKGROUND: The enzyme cellobiose dehydrogenase (CDH) can be used to oxidize lactose to lactobionic acid. As Sclerotium rolfsii is known to be a good producer of CDH, the aim of this paper was to simplify its production and secondly to systematically study its purification aiming for a high yield. Two preservation methods (freezing and freeze-drying) and the influence of several protectants were investigated. RESULTS: Production of cellobiose dehydrogenase was optimized leading to a more simplified medium composition. Purification of the enzyme was evaluated by determining breakthrough profiles on different ion exchange (IEX) and hydrophobic interaction (HIC) materials with regard to buffer composition. Highest purification with an acceptable loss during the capture step using IEX was obtained with a Q Sepharose XL medium and a 100 mM sodium acetate buffer at pH 4.5. Subsequent purification using hydrophobic interaction chromatography was done at 1.1 M ammonium sulfate concentration. Purification was moderate, yielding a specific activity of 11.9 U/mg (56% yield). However, as could be shown in a preliminary experiment, purity of the obtained enzyme solution was sufficient for its intended use to oxidize lactose to lactobionic acid. Various sugars and sugar alcohols were investigated to study their protective effect during lyophilisation and freezing at -20 °C. Glucose and lactulose could be identified to have a high lyoprotective effect while loss of enzyme activity was high (77%) when using no additives. CONCLUSION: By simplifying the cultivation medium of Sclerotium rolfsii, the costs of cellobiose dehydrogenase production could be reduced. Simultaneously, CDH production was increased by 21%. The production of lactobionic acid from lactose is possible using partially purified and unpurified enzyme. Storage at -20 °C using 50% (w/v) glycerol was considered to be most suited for preservation of the enzyme.

Just in time-selection: A rapid semiautomated SELEX of DNA aptamers using magnetic separation and BEAMing.

A semiautomated two-step method for in vitro selection of DNA aptamers using magnetic separation and solid-phase emulsion polymerase chain reaction has been developed. The application of a magnetic separator allows the simultaneous processing of up to 12 SELEXs (systematic evolution of ligands by exponential enrichment) with different targets or buffer conditions. Using a magnetic separator and covalent target immobilization on magnetic beads, the selection process was simplified and the substeps of aptamer/target incubation, washing, and elution of the aptamers were merged into one automated procedure called "FISHing". Without further processing the resulting FISHing eluates are suitable for BEAMing (beads, emulsion, amplification, and magnetics), which includes the amplification by emPCR (emulsion polymerase chain reaction) and strand separation by the implementation of covalently immobilized reverse primers on magnetic beads. The novel selection process has been proved and validated by selecting and characterization of aptamers to the wine fining agent lysozyme.