Xylanases and high-degree wet milling improve soluble dietary fibre content in liquid oat base.

The growth of plant-based food and drink substitutes has led to increased interest in oat-based milk substitute as a dairy milk alternative. Conventional liquid oat base (LOB) production results in a fibre-rich insoluble by-product and loss of valuable macronutrients. This study investigates the use of xylanase enzymes to release insoluble arabinoxylan (AX) fibre and employs different degrees of milling in the LOB manufacturing process, with the aim to reduce insoluble waste and simultaneously increase soluble dietary fibre in oat-based milk substitutes. The combination of decreased mill gap space from 1 to 0.05 mm and addition of GH10 xylanase, resulted in a homogenous LOB product and solubilization of all available AX. Potential prebiotic arabinoxylooligosaccharides of DP3-7 from GH10 hydrolysis were identified using HPAEC-PAD and MS analysis. These findings demonstrate the value of utilizing xylanases and fine-milling in LOB manufacturing, offering a sustainable approach to maximize health benefits of oat-based beverages.

Evaluation of Polytyramine Film and 6-Mercaptohexanol Self-Assembled Monolayers as the Immobilization Layers for a Capacitive DNA Sensor Chip: A Comparison.

The performance of a biosensor is associated with the properties of an immobilization layer on a sensor chip. In this study, gold sensor chips were modified with two different immobilization layers, polytyramine film and 6-mercaptohexanol self-assembled monolayer. The physical, electrochemical and analytical properties of polytyramine film and mercaptohexanol self-assembled monolayer modified gold sensor chips were studied and compared. The study was conducted using atomic force microscopy, cyclic voltammetry and a capacitive DNA-sensor system (CapSenze™ Biosystem). The results obtained by atomic force microscopy and cyclic voltammetry indicate that polytyramine film on the sensor chip surface possesses better insulating properties and provides more spaces for the immobilization of the capture probe than a mercaptohexanol self-assembled monolayer. A capacitive DNA sensor hosting a polytyramine single-stranded DNA-modified sensor chip displayed higher sensitivity and larger signal amplitude than that of a mercaptohexanol single-stranded DNA-modified sensor chip. The linearity responses for polytyramine single-stranded DNA- and mercaptohexanol single-stranded DNA-modified sensor chips were obtained at log concentration ranges, equivalent to 10-12 to 10-8 M and 10-10 to 10-8 M, with detection limits of 4.0 × 10-13 M and 7.0 × 10-11 M of target complementary single-stranded DNA, respectively. Mercaptohexanol single-stranded DNA- and polytyramine single-stranded DNA-modified sensor chips exhibited a notable selectivity at an elevated hybridization temperature of 50 °C, albeit the signal amplitudes due to the hybridization of the target complementary single-stranded DNA were reduced by almost 20% and less than 5%, respectively.

Novel multiplex capacitive sensor based on molecularly imprinted polymers: A promising tool for tracing specific amphetamine synthesis markers in sewage water.

The development of a sensing system for amphetamine (AMP), N-formyl amphetamine (NFA), and benzyl methyl ketone (BMK) in sewage is a strict requirement for enabling the on-site detection and tracing of the consumption of AMP, and the production and/or transportation of these target analytes. The present research is therefore devoted to the development of an on-site capacitive sensing system, based on molecularly imprinted polymers (MIPs) as recognition elements. To this end, the commercially available CapSenze capacitive sensor system was miniaturized by implementing an application-specific integrated circuit (ASIC), dedicated to the bias and read-out of the chemical sensor. MIPs towards AMP were purchased, whereas the ones towards NFA and BMK were synthesized in house. Gold transducers, consisting of six working electrodes with their corresponding reference electrodes and one common auxiliary electrode, were designed together with a flow cell to enable analyses. The applied water samples were filtered through a 20 micron filter before application in the sensors' flow cell. The limits of detection in filtered sewage water were determined to be 25 µM for NFA and BMK and 50 µM for AMP. The overall performance of the sensing system was tested by analysis of blind-coded sewage samples, provided by legal authorities. To the best of our knowledge, this is the first research presenting multiplex MIP-based detection of amphetamine synthesis markers using a capacitive sensor, miniaturized via ASIC technology. The presented technique is undoubtedly a potential solution for any analysis requiring constant reliable on-site monitoring of a substance of interest.

A Sensitive Capacitive Biosensor for Protein a Detection Using Human IgG Immobilized on an Electrode Using Layer-by-Layer Applied Gold Nanoparticles.

A capacitive biosensor for the detection of protein A was developed. Gold electrodes were fabricated by thermal evaporation and patterned by photoresist photolithography. A layer-by-layer (LbL) assembly of thiourea (TU) and HAuCl4 and chemical reduction was utilized to prepare a probe with a different number of layers of TU and gold nanoparticles (AuNPs). The LbL-modified electrodes were used for the immobilization of human IgG. The binding interaction between human IgG and protein A was detected as a decrease in capacitance signal, and that change was used to investigate the correlation between the height of the LbL probe and the sensitivity of the capacitive measurement. The results showed that the initial increase in length of the LbL probe can enhance the amount of immobilized human IgG, leading to a more sensitive assay. However, with thicker LbL layers, a reduction of the sensitivity of the measurement was registered. The performance of the developed system under optimum set-up showed a linearity in response from 1 × 10-16 to 1 × 10-13 M, with the limit detection of 9.1 × 10-17 M, which could be interesting for the detection of trace amounts of protein A from affinity isolation of therapeutic monoclonal antibodies.

Rapid detection of mecA gene of methicillin-resistant Staphylococcus aureus by a novel, label-free real-time capacitive biosensor.

This work presents a rapid, selective and sensitive automated sequential injection flow system with a capacitive biosensor for detection of the mecA gene (the model chosen for this study), which emerges from methicillin-resistant Staphylococcus aureus. A DNA-based 25-mer capture probe was immobilized on the surface of a gold electrode which was integrated in the capacitive sensor system. A constant current pulse was applied and the resulting capacitance was measured. Injection of the target DNA sample to the sensor surface induced hybridization to occur between the target and the complementary sequence, which resulted in a shift in the measured capacitance (ΔC). The ΔC was directly proportional to the concentrations of the applied target probe with linearity ranging from 10-12 to 10-7 M. The biosensor had a detection limit of 6.0 × 10-13 M and a recovery of 95 % of the mecA gene when spiked in human saliva. The biosensor showed a promising selectivity. It could clearly discriminate single-base, two-base and twelve-base mismatch probes with a decrease in the signal strength by 13 %, 26 %, and 89 %, respectively relative to the signal strength of the complementary target probe. There was no significant signal observed for the non-complementary probe. The biosensor-chip could be re-used for more than 12 cycles with residual capacity of 94.5 ± 4.3 % and a RSD of 4.6 % by regenerating the biosensor-chip with a solution of 50 mM NaOH.

Development of an automated flow-based spectrophotometric immunoassay for continuous detection of zearalenone.

Considering the widespread contaminations of food products with mycotoxins, it is important to develop, robust, time- and cost-effective detection methods. We developed and optimized an immunoassay using a continuous flow system for the detection of zearalenone (ZEN). The assay was performed in a flow mode using an automated sequential injection system. Time for an assay cycle was 18 Min. Under optimal conditions, the limit for quantification for ZEN was 0.40 µg L-1 with a linear dependency between concentration and signal amplitude between 0.10 and 10 µg L-1 . The assay proved to be robust and reliable with 13% relative standard deviation between measurements. By dissociating the antigen-antibody complex using a regeneration solution, we showed 50 times reusability of the immobilized antibodies without affecting the antigen-binding properties.

Capacitive Sensor to Monitor Enzyme Activity by Following Degradation of Macromolecules in Real Time.

A capacitive sensor was developed to analyze the presence and enzymatic activity of a model protease from standard solutions by following the degradation of the substrate in real time. The enzyme was chosen based on its specific digestion of the hinge region of immunoglobulin G (IgG). Real-time enzyme activity was monitored by measuring the change in capacitance (∆C) based on the release of IgG fragments after enzymatic digestion by the enzyme. The results indicated that the developed capacitive system might be used successfully for label-free and real-time monitoring of enzymatic activity of different enzymes in a sensitive, rapid, and inexpensive manner in biotechnological, environmental, and clinical applications.

Antibody immobilization strategy for the development of a capacitive immunosensor detecting zearalenone.

A highly sensitive flow-injection capacitive immunosensor was developed for detection of the mycotoxin zearalenone (ZEN). Different strategies for immobilization of an anti-ZEN antibody on the surface of a gold electrode, i.e. polytyramine or self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (3-MPA) and lipoic acid (LA), were used and their performances were compared. The LA- and 3-MPA-based systems showed broad linear ranges for ZEN determination, i.e. from 0.010 nM to 10 nM and from 0.020 nM to 10 nM, respectively. Under optimal conditions, the LA-based immunosensor was capable of performing up till 13 regeneration-interaction cycles (with use of glycine HCl, pH 2.4) with a limit of detection (LOD) of 0.0060 nM, equivalent to 1.9 pg mL-1. It also demonstrated a good inter-assay precision (RSD < 10%). However, the tyramine-based capacitive immunosensor showed a bad repeatability (only 4 regeneration-interaction cycles were possible) and inter-assay precision (RSD > 15%) which did not allow sensitive and precise measurements. The LA-based method was compared with a direct ELISA. These results demonstrated that the label-free developed capacitive immunosensor had a better sensitivity and shorter analysis time in comparison with the direct microwell-plate format.

Capacitive Saccharide Sensor Based on Immobilized Phenylboronic Acid with Diol Specificity.

A capacitive sensor for saccharide detection is described in this study. The detection is based on selective interaction between diols and aminophenylboronic acid (APBA) immobilized on a gold electrode. Glucose, fructose, and dextran (MW: 40 kDa) were tested with the system over wide concentration ranges (1.0 x 10-8 M - 1.0 x 10-3 M for glucose, 1.0 x 10-8 M - 1.0 x 10-2 M for fructose and 1.0 x 10-10 M - 1.0 x 10-5 M for dextran). The limits of detection (LODs) were 0.8 nM for glucose, 0.6 nM for fructose, and 13 pM for dextran. These data were comparable to the others reported previously. In order to demonstrate glycoprotein detection with the same sensor, human immunoglobulin G (IgG) as well as horseradish peroxidase were used as model analytes. The sensor responded to IgG in the concentration range of 1.0 x 10-13 M - 1.0 x 10-7 M with a LOD value of 16 fM. The performance of the assay of peroxidase was compared to a spectrophotometric assay by determining the enzymatic activity of a captured analyte. The results showed that the method might be useful for label-free, fast, and sensitive detection of saccharides as well as glycoproteins over a wide concentration range.

A novel capacitive sensor based on molecularly imprinted nanoparticles as recognition elements.

Molecularly Imprinted Polymers (MIPs) are synthetic receptors capable of selective binding to their target (template) molecules and, hence, are used as recognition elements in assays and sensors as a replacement for relatively unstable enzymes and antibodies. Herein, we describe a manufacturing-friendly protocol for integration of MIP nanoparticles (nanoMIPs) with a (label-free) capacitive sensor. The nanoMIPs were produced by solid-phase synthesis for two templates with different sizes and properties, including a small molecule tetrahydrocannabinol (THC) and a protein (trypsin). NanoMIPs were deposited on the surface of the sensor and the change in capacitance (ΔC) upon binding of the target was measured. The significant improvement in the selectivity and limit of detection (one order of magnitude compared to previously used MIP microparticles) can be attributed to their increased surface-to-volume ratio and higher specificity of the nanoMIPs produced by the solid-phase method. The methodology described is also compatible with common sensor fabrication approaches, as opposed to methods involving in situ MIP polymerisation. The proposed sensor shows high selectivity, fast sensor response (45 min including injection, regeneration and re-equilibration with running buffer), and straightforward data analysis, which makes it viable for label-free monitoring in real-time. The set of targets assessed in this manuscript shows the general applicability of the biosensor platform.

Highly sensitive detection and quantification of the secreted bacterial benevolence factor RoxP using a capacitive biosensor: A possible early detection system for oxidative skin diseases.

The impact of the microbiota on our health is rapidly gaining interest. While several bacteria have been associated with disease, and others being indicated as having a probiotic effect, the individual biomolecules behind these alterations are often not known. A major problem in the study of these factors in vivo is their low abundance in complex environments. We recently identified the first secreted bacterial antioxidant protein, RoxP, from the skin commensal Propionibacterium acnes, suggesting its relevance for maintaining the redox homeostasis on the skin. In order to study the effect, and prevalence, of RoxP in vivo, a capacitive biosensor with a recognition surface based on molecular imprinting was used to detect RoxP on skin in vivo. In vitro analyses demonstrated the ability to detect and quantify RoxP in a concentration range of 1 x 10-13 M to 1 x 10-8 M from human skin swabs; with a limit of detection of 2.5 x 10-19 M in buffer systems. Further, the biosensor was highly selective, not responding to any other secreted protein from P. acnes. Thus, it was possible to demonstrate the presence, and quantity, of RoxP on human skin. Therefore, the developed biosensor is a very promising tool for the detection of RoxP from clinical samples, offering a rapid, cost-effective and sensitive means of detecting low-abundant bacterial proteins in vivo in complex milieus.

Electrochemical Flow-ELISA for Rapid and Sensitive Determination of Microcystin-LR Using Automated Sequential Injection System.

An amperometric immunoanalysis system based on monoclonal antibodies immobilized on Sepharose beads and packed into a micro-immunocolumn was developed for the quantification of microcystin-LR. Microcystin-LR (MCLR) was used as a reference microcystin variant. Inside the immunocolumn, free microcystins and microcystin-horseradish peroxidase (tracer) were sequentially captured by the immobilized antibodies, and the detection was performed electrochemically using Super AquaBlue ELISA substrate 2,2'-azinobis(3-ethylbenzothiazoline-sulfonic acid) (ABTS). The ABTS●+ generated by enzymatic oxidation of ABTS was electrochemically determined at a carbon working electrode by applying a reduction potential set at 0.4 V versus Ag/AgCl reference electrode. The peak current intensity was inversely proportional to the amount of analyte bound to the immunocolumn. The amperometric flow-ELISA system, which was automatically controlled through the CapSenzeTM (Lund, Sweden) computer software, enabled determination of MCLR as low as 0.01 µg/L. The assay time was very short (20 min for one assay cycle). In addition, the electrochemical signals were not significantly affected by possible interferences which could be present in the real samples. Along with the simplicity of automation, this makes the developed method a promising tool for use in water quality assessment.

Whole cell based microcontact imprinted capacitive biosensor for the detection of Escherichia coli.

In this study, a label-free, selective and sensitive microcontact imprinted capacitive biosensor was developed for the detection of Escherichia coli. The recognition of E. coli was successfully performed by this sensor prepared with the combination of microcontact imprinting method and capacitive biosensor technology. After preparation of bacterial stamps, microcontact-E. coli imprinted gold electrodes were generated using an amino acid based recognition element, N-methacryloyl-L-histidine methylester (MAH), 2-Hydroxyethyl methacrylate (HEMA) as monomers and ethyleneglycol dimethacrylate (EGDMA) as crosslinker under UV-polymerization. Real-time E. coli detection experiments were carried out within the range of 1.0×102-1.0×107CFU/mL. The unique combination of these two techniques provides selective detection with a detection limit of 70CFU/mL. The designed capacitive sensor has high selectivity and was able to distinguish E. coli when present together with competing bacterial strains which are known to have similar shape. In addition, the prepared sensor has the ability to detect E. coli with a recovery of 81-97% in e.g. river water.

A sensitive and real-time assay of trypsin by using molecular imprinting-based capacitive biosensor.

Use of a highly sensitive, selective capacitive biosensor is reported for label-free, real-time, easy and rapid detection of trypsin by using the microcontact imprinting method. Real-time trypsin detection was performed with trypsin-imprinted (trypsin-MIP) capacitive electrodes using standard trypsin solutions in the concentration range of 1.0×10(-13)-1.0×10(-7)M with a detection limit of 3.0×10(-13)M. Selectivity and cross-reactivity of the system were tested by using competing proteins including chymotrypsin (chy), bovine serum albumin (BSA), lysozyme (lyz) and cytochrome c (cyt c) in singular and competitive manner and the selectivity of the system was determined with the selectivity coefficients of approximately 705.1, 6.5, 6.4 and 5.1 for chy, BSA, lyz and cyt c, respectively. The trypsin-MIP capacitive electrode was used for ~80 assays during 2 months and retained its binding property during all that time with a decrease of approximately 2.3% in the signal amplitude. In the last step, trypsin activity was measured by using Nα-Benzoyl-D, l-arginine 4-nitroanilide hydrochloride (BAPNA) as the substrate with spectrophotometer at 410nm. The trypsin activity was measured as 9mU/mL by spectrophotometer while the amount of captured enzyme calculated from the capacitive system was 7.9mU/mL which shows the correlation between two methods. From the comparison it is obvious that the new method is an attractive alternative for assaying trypsin and the developed capacitive system might be used successfully to monitor label-free, real-time enzymatic activity of different proteases in a sensitive, rapid, cost-effective manner for different applications.

The Bacterial (Vibrio alginolyticus) Production of Tetrodotoxin in the Ribbon Worm Lineus longissimus-Just a False Positive?

We test previous claims that the bacteria Vibrio alginolyticus produces tetrodotoxin (TTX) when living in symbiosis with the nemertean Lineus longissimus by a setup with bacteria cultivation for TTX production. Toxicity experiments on the shore crab, Carcinus maenas, demonstrated the presence of a paralytic toxin, but evidence from LC-MS and electrophysiological measurements of voltage-gated sodium channel-dependent nerve conductance in male Wistar rat tissue showed conclusively that this effect did not originate from TTX. However, a compound of similar molecular weight was found, albeit apparently non-toxic, and with different LC retention time and MS/MS fragmentation pattern than those of TTX. We conclude that C. maenas paralysis and death likely emanate from a compound <5 kDa, and via a different mechanism of action than that of TTX. The similarity in mass between TTX and the Vibrio-produced low-molecular-weight, non-toxic compound invokes that thorough analysis is required when assessing TTX production. Based on our findings, we suggest that re-examination of some published claims of TTX production may be warranted.

Bioimprinting as a tool for the detection of aflatoxin B1 using a capacitive biosensor.

A strategy for the detection of aflatoxin B1 using a capacitive biosensor has been studied. The use of proteins for the generation of sites with high specificity against aflatoxin B1 are produced via bioimprinting. This technique has become a tool for the detection of aflatoxin B1 using a capacitive biosensor. The results demonstrate the ability to generate specific interactions with aflatoxin B1 with a linear relation between signals registered and log concentration of the target aflatoxin in the concentration range of 3.2 × 10-6 to 3.2 × 10-9 M when using ovalbumin as framework for the bioimprinting.

Real-time prostate-specific antigen detection with prostate-specific antigen imprinted capacitive biosensors.

Prostate specific antigen (PSA) is a valuable biomarker for early detection of prostate cancer, the third most common cancer in men. Ultrasensitive detection of PSA is crucial to screen the prostate cancer in an early stage and to detect the recurrence of the disease after treatment. In this report, microcontact-PSA imprinted (PSA-MIP) capacitive biosensor chip was developed for real-time, highly sensitive and selective detection of PSA. PSA-MIP electrodes were prepared in the presence of methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker via UV polymerization. Immobilized Anti-PSA antibodies on electrodes (Anti-PSA) for capacitance measurements were also prepared to compare the detection performances of both methods. The electrodes were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV) and real-time PSA detection was performed with standard PSA solutions in the concentration range of 10 fg mL(-1)-100 ng mL(-1). The detection limits were found as 8.0 × 10(-5) ng mL(-1) (16 × 10(-17) M) and 6.0 × 10(-4) ng mL(-1) (12 × 10(-16) M) for PSA-MIP and Anti-PSA electrodes, respectively. Selectivity studies were performed against HSA and IgG and selectivity coefficients were calculated. PSA detection was also carried out from diluted human serum samples and finally, reproducibility of the electrodes was tested. The results are promising and show that when the sensitivity of the capacitive system is combined with the selectivity and reproducibility of the microcontact-imprinting procedure, the resulting system might be used successfully for real-time detection of various analytes even in very low concentrations.

Affinity sensor based on immobilized molecular imprinted synthetic recognition elements.

An affinity sensor based on capacitive transduction was developed to detect a model compound, metergoline, in a continuous flow system. This system simulates the monitoring of low-molecular weight organic compounds in natural flowing waters, i.e. rivers and streams. During operation in such scenarios, control of the experimental parameters is not possible, which poses a true analytical challenge. A two-step approach was used to produce a sensor for metergoline. Submicron spherical molecularly imprinted polymers, used as recognition elements, were obtained through emulsion polymerization and subsequently coupled to the sensor surface by electropolymerization. This way, a robust and reusable sensor was obtained that regenerated spontaneously under the natural conditions in a river. Small organic compounds could be analyzed in water without manipulating the binding or regeneration conditions, thereby offering a viable tool for on-site application.

Screening of self-assembled monolayer for aflatoxin B1 detection using immune-capacitive sensor.

A capacitive biosensor was used for detection of aflatoxin B1. Two different methods for cleaning gold electrodes were evaluated using cyclic voltammetry in the presence of ferricyanide as redox couple. The methods involve use of a sequence of cleaning steps avoiding the use of Piranha solution and plasma cleaner. Anti-aflatoxin B1 was immobilized on self-assembled monolayers (SAM). The immune-capacitive biosensor is able to detect aflatoxin B1 concentrations in a linear range of 3.2 × 10-12 M to 3.2 × 10-9 M when thiourea was used to form the SAM; 3.2 × 10-9 M to 3.2 × 10-7 M when thioctic acid was used. When the gold surface was isolated with tyramine-electropolymerization linear ranges of 3.2 × 10-13 M to 3.2 × 10-7 M and 3.2 × 10-9 M to 3.2 × 10-7 M where obtained, respectively. The results obtained show the difference in linear range, limit of detection, and limit of quantification when different self-assembled monolayers are used for aflatoxin B1 detection.

A capacitive DNA sensor-based test for simple and sensitive analysis of antibiotic resistance in field setting.

To meet urgent needs for solving serious worldwide drug-resistance problems, a sensitive label-free capacitive sensor developed in our group was investigated as a tool to be applied in the field of antibiotic resistance genotyping, for instance the detection of ampicillin resistance gene (ampR). Proof-of-concept data demonstrated its detection sensitivity of pico-molar without any signal amplification step and a dynamic range of at least three orders of magnitude. The detection limits of less than 1 pM for the single-stranded ampR oligonucleotide and 4 pM for the double-stranded target can reliably be achieved after only 2.5 min sample reaction. Reusability of the probe-functionalized disposable electrode was investigated by comparing different regeneration solutions; mix of 25 mM NaOH/30% formamide was employed to regenerate the electrode for at least six cycles without significant loss of sensing ability. Assay is performed automatically and result is retrieved in 20 min. The developed sensitive genotyping tool is expected to provide simple, fast and affordable screening for monitoring spread of antibiotic resistances, which is suitable for testing in field setting.