Flow-through Biosensor Research Articles

A Flow-Through Biosensor System Based on Pillar[3]Arene[2]Quinone and Ferrocene for Determination of Hydrogen Peroxide and Uric Acid

Simple and reliable electrochemical sensors are highly demanded in medicine and pharmacy for the fast determination of metabolites and biomarkers of diseases. In this work, a flow-through biosensor system was developed on the base of a screen-printed carbon electrode modified with pillar[3]arene[2]quinone and ferrocene implemented in carbon black. The modification was performed in a single step and resulted in the formation of a stable layer with good operation characteristics. Uricase was immobilized on the inner walls of a replaceable reactor by carbodiimide binding. A flow-through cell was manufactured by 3D printing from poly(lactic acid). The flow-through system was first optimized on the hydrogen peroxide assay and then used for the determination of 1 nM–0.1 mM uric acid (limit of detection 0.3 nM, 20 measurements per hour). Implementation of ferrocene resulted in a synergetic increase in the cathodic current of H2O2 reduction measured by flow switching in chronoamperometric mode. The developed system was tested on the determination of uric acid in artificial urine and Ringer–Locke solution and showed a recovery rate of 96–112%. In addition, the possibility of determination of H2O2 in commercial disinfectants was shown. Easy assembly, fast and reliable signal and low consumption of the reagents make the system developed attractive for routine clinical analysis of metabolites.

Flow-Through Amperometric Biosensor System Based on Functionalized Aryl Derivative of Phenothiazine and PAMAM-Calix-Dendrimers for the Determination of Uric Acid.

A flow-through biosensor system for the determination of uric acid was developed on the platform of flow-through electrochemical cell manufactured by 3D printing from poly(lactic acid) and equipped with a modified screen-printed graphite electrode (SPE). Uricase was immobilized to the inner surface of a replaceable reactor chamber. Its working volume was reduced to 10 μL against a previously reported similar cell. SPE was modified independently of the enzyme reactor with carbon black, pillar[5]arene, poly(amidoamine) dendrimers based on the p-tert-butylthiacalix[4]arene (PAMAM-calix-dendrimers) platform and electropolymerized 3,7-bis(4-aminophenylamino) phenothiazin-5-ium chloride. Introduction of the PAMAM-calix-dendrimers into the electrode coating led to a fivefold increase in the redox currents of the electroactive polymer. It was found that higher generations of the PAMAM-calix-dendrimers led to a greater increase in the currents measured. Coatings consisted of products of the electropolymerization of the phenothiazine with implemented pillar[5]arene and PAMAM-calix-dendrimers showing high efficiency in the electrochemical reduction of hydrogen peroxide that was formed in the enzymatic oxidation of uric acid. The presence of PAMAM-calix-dendrimer G2 in the coating increased the redox signal related to the uric acid assay by more than 1.5 times. The biosensor system was successfully applied for the enzymatic determination of uric acid in chronoamperometric mode. The following optimal parameters for the chronoamperometric determination of uric acid in flow-through conditions were established: pH 8.0, flow rate 0.2 mL·min-1, 5 U of uricase per reactor. Under these conditions, the biosensor system made it possible to determine from 10 nM to 20 μM of uric acid with the limit of detection (LOD) of 4 nM. Glucose (up to 1 mM), dopamine (up to 0.5 mM), and ascorbic acid (up to 50 μM) did not affect the signal of the biosensor toward uric acid. The biosensor was tested on spiked artificial urine samples, and showed 101% recovery for tenfold diluted samples. The ease of assembly of the flow cell and the low cost of the replacement parts make for a promising future application of the biosensor system in routine clinical analyses.

Electrochemical flow-through biosensors based on microfiber enzymatic filter discs placed at printed electrodes

A new type of electrochemical biosensors in a flow injection system with printed electrodes were developed and tested. A filter disc (7 mm diameter) with immobilized enzyme was placed at the printed electrode. This conception combines the advantages of biosensors with a bioreceptor at the electrode surface and systems with spatially separated enzymatic and detection parts. Filters of different composition (glass, quartz, and cellulose), thickness, porosity, and ways of binding enzyme to their surface were tested. Only covalent bonds throughout a filter-aminosilane-glutaraldehyde-enzyme chain ensured a long-time and reproducible biosensor response. The developed method of biosensor preparation has been successfully applied to enzymes glucose oxidase, laccase and choline oxidase. The dependences of peak current on detection potential, flow rate, injection volume, analyte concentration as well as biosensor lifetime and reproducibility were investigated for glucose oxidase biosensor. The sensitivity of measurements was two or more times higher than that of biosensor with a mini-reactor filled by powder with immobilized enzyme. The developed biosensor with laccase was tested by determining dopamine in the pharmaceutical infusion product Tensamin®. Results of the analysis (40.0 ± 0.7 mg mL−1, SD = 0.8 mg mL−1, RSD = 1.85 %, N = 11) show a good agreement with the manufacturer's declared value.

Flow-Through Acetylcholinesterase Sensor with Replaceable Enzyme Reactor.

Fast and reliable determination of enzyme inhibitors are of great importance in environmental monitoring and biomedicine because of the high biological activity and toxicity of such species and the necessity of their reliable assessment in many media. In this work, a flow-through biosensor has been developed and produced by 3D printing from poly(lactic acid). Acetylcholinesterase from an electric eel was immobilized on the inner walls of the reactor cell. The concentration of thiocholine formed in enzymatic hydrolysis of the substrate was monitored amperometrically with a screen-printed carbon electrode modified with carbon black particles, pillar[5]arene, electropolymerized Methylene blue and thionine. In the presence of thiocholine, the cathodic current at −0.25 V decreased because of an alternative chemical reaction of the macrocycle. The conditions of enzyme immobilization and signal measurements were optimized and the performance of the biosensor was assessed in the determination of reversible (donepezil, berberine) and irreversible (carbofuran) inhibitors. In the optimal conditions, the flow-through biosensor made it possible to determine 1.0 nM–1.0 μM donepezil, 1.0 μM–1.0 mM berberine and 10 nM to 0.1 μM carbofuran. The AChE biosensor was tested on spiked samples of artificial urine for drugs and peanuts for carbofuran. Possible interference of the sample components was eliminated by dilution of the samples with phosphate buffer. Easy mounting, low cost of replaceable parts of the cell and satisfactory analytical and metrological characteristics made the biosensor a promising future application as a point-of-care or point-of-demand device outside of a chemical laboratory.

Simultaneous monitoring of sweat lactate content and sweat secretion rate by wearable remote biosensors

We report on the simultaneous monitoring of sweat lactate concentration and sweat secretion rate. For this aim lactate oxidase-Prussian Blue enzyme-nanozyme type lactate biosensors were elaborated. The use of siloxane-perfluorosulfonated ionomer composite membrane for enzyme-nanozyme immobilization results in the biosensor displaying flux independence in the whole range of physiological sweat secretion rates (0.025–2 μl cm-2 min-1). On the contrary, current response of the biosensor based on solely siloxane membranes becomes saturated at physiological sweat lactate concentration, depending mostly on the flow rate. Accordingly, for simultaneous monitoring of sweat lactate concentration and its secretion rate both flow-through biosensors were integrated with high-accuracy wearable electronic devices allowing real-time remote monitoring. As found, during exhaustive physical exercise sweat secretion rate and lactate content are independent of each other, thus, confirming that this excretory liquid is suitable for non-invasive diagnostics.

Flow-Through Electrochemical Biosensor with a Replaceable Enzyme Reactor and Screen-Printed Electrode for the Determination of Uric Acid and Tyrosine

A flow-through biosensor has been developed and manufactured by 3D printing from poly(lactic acid). Uricase or tyrosinase were immobilized on the inner walls of the reactor cell and the concentrations of the reaction products, i.e., hydrogen peroxide in the case of uricase and quinone product of the tyrosine oxidation in the case of tyrosinase, were monitored in the amperometric mode. The screen-printed electrode modified with carbon black, pillar[5]arene and electropolymerized thionine and methylene blue was separated from the enzyme layer by 0.1 mm gap and used as a biosensor transducer. The components of the modifier layer exerted a synergetic effect and increased the cathodic current up to seven times compared to electrodes modified with individual components, increasing the sensitivity of the analysis. In the optimal conditions, biosensor allowed the determination of 1 × 10−7 to 1 × 10−5 M uric acid and 2 × 10−7 to 1 × 10−5 M tyrosine. The biosensors analyzed spiked samples of blood serum and urine and provided recoveries from 95 to 110%. Easy mounting of the flow-through cell and low cost of the replaceable parts make promising future application of the biosensor for routine clinical assays.

Lateral porous silicon interferometric transducer for on-chip flow-through sensing applications

Most porous silicon-based interferometric sensors targeting biosensing applications consist of vertical porous silicon layers created into a silicon wafer by electrochemical anodization and operate in a flow-over configuration. In this work, we present an alternative porous silicon interferometer based on porous silicon with horizontally oriented pores. This architecture permits the integration of flow-through porous silicon membranes within planar microfluidics. Fourier-transform infrared spectroscopy was used to obtain interference spectra from fabricated lateral porous silicon membranes and red shifts were observed upon filling microfluidic chips integrating the porous membranes with solvents of higher optical indices. This work proves that lateral porous silicon membranes are typical Fabry-Pérot interferometers with a sensitivity of more than 150 nm/RIU and a limit of detection less than 10−3 RIU, that is comparable to vertical porous silicon layers. Moreover, we have conducted simulation studies showing that the addition of Bragg mirrors on the membranes results in spectra with narrower fringes and lateral porous silicon interferometers with improved performances. After appropriate biofunctionalization of the porous silicon surface, lateral porous silicon membrane interferometers should offer alternative solutions for the development of porous silicon flow-through biosensors monolithically integrated on-chip.

Performance Comparison of Flow-Through Optofluidic Biosensor Designs.

Optofluidic flow-through biosensors are being developed for single particle detection, particularly as a tool for pathogen diagnosis. The sensitivity of the biosensor chip depends on design parameters, illumination format (side vs. top), and flow configuration (parabolic, two- and three-dimensional hydrodynamic focused (2DHF and 3DHF)). We study the signal differences between various combinations of these design aspects. Our model is validated against a sample of physical devices. We find that side-illumination with 3DHF produces the strongest and consistent signal, but parabolic flow devices process a sample volume more quickly. Practical matters of optical alignment are also discussed, which may affect design choice.

Numerical Modeling for Sensitive and Rapid Molecular Detection by Membrane-Based Immunosensors.

Rapid, simple, and sensitive point-of-care testing (POCT) has attracted attention in recent years due to its excellent potential for early disease diagnosis and health monitoring. The flow-through biosensor design is a candidate for POCT that utilizes the small-sized pores of a porous membrane as a recognition space where it emits a signal comparable to that of a conventional enzyme-linked immunosorbent assay within 35 min of detection time. In this paper, we present a numerical model for this immunosensing technology to systematically design an improved recognition system. The model considers mass transfer into the pore (convection and diffusion), the kinetics between the immobilized receptor and the target molecule, and the flow conditions, successfully leading to a bottleneck step (capture of secondary antibody) in sandwich-type detection. Our simulation results also show that this problem can be solved by adopting both appropriate receptors and analytical conditions. Eventually, the requirements to achieve the sensitivity required for POCT were fulfilled, which will allow for further development of immunosensing devices for disease detection.

Noninvasive monitoring of diabetes and hypoxia by wearable flow-through biosensors

Abstract Noninvasively collected sweat via clinically relevant procedure can be used for assessing the human condition, particularly for monitoring of diabetes and hypoxia. Positive correlations in variation rates, claimed as sufficient requirements for diagnostics, between glucose and lactate concentrations in sweat and the corresponding values in blood are shown. Continuous monitoring of human condition is possible only with the use of flow-through wearable devices providing a delivery of sweat to the biosensor almost immediately after excretion. Owing to both low sweat glucose content and inactivation of platinum by sweat components, the use of Prussian blue–based biosensors for wearable devices is preferable. Operation of the latter in power generation mode allows significant noise reduction and clear readout of the signal. Evaluating blood glucose through sweat analysis upon glucose tolerance test, we clearly show that humans can actually be monitored reliably via noninvasive approach.

An optofluidic metasurface for lateral flow-through detection of breast cancer biomarker

The rapid growth of point-of-care tests demands for biosensors with high sensitivity and small size. This paper demonstrates an optofluidic metasurface that combines silicon-on-insulator (SOI) nanophotonics and nanofluidics to realize a high-performance, lateral flow-through biosensor. The metasurface is made of a periodic array of silicon nanoposts on an SOI substrate, and functionalized with specific receptor molecules. Bonding of a polydimethylsiloxane slab directly onto the surface results in an ultracompact biosensor, where analyte solutions are restricted to flow only in the space between the nanoposts. No flow exists above the nanoposts. This sensor design overcomes the issue with diffusion-limited detection of many other biosensors. The lateral flow-through feature, in conjunction with high-Q resonance modes associated with optical bound states of the metasurface, offers an improved sensitivity to subtle molecule-bonding induced changes in refractive index. The device exhibits a resonance mode around 1550 nm wavelength and provides an index sensitivity of 720 nm/RIU. Biosensing is conducted to detect the epidermal growth factor receptor 2 (ErbB2), a protein biomarker for early-stage breast cancer screening, by monitoring resonance wavelength shifts in response to specific analyte-ligand binding events at the metasurface. The limit of detection of the device is 0.7 ng mL−1 for ErbB2.

Determination of Penicillins in Milk by a Dual-Optrode Biosensor

ABSTRACTPenicillins are the most frequently found antibiotic residues in milk, as they are commonly used for the treatment of bacterial infections in cows. In the present study, we introduce a method for the rapid detection of penicillin residues in raw milk based on the determination of glucose concentration in milk with a dual flow-through biosensor. The molar concentration of glucose in milk is typically over 500 times lower than the concentration of lactose and is highly dependent on the rate of lactose hydrolysis, which is catalyzed by β-galactosidase. Glucose concentrations in milk change with variation in the β-galactosidase activity. β-Galactosidase is an enzyme produced in the microbiota in milk and its activity is inhibited by benzylpenicillin. Spiking milk with benzylpenicillin lowers glucose concentrations in comparison to high-quality milk after short storage intervals. The presence of penicillin in the milk of treated animals resulted in decreased glucose concentrations in comparison with high-quality milk that contained no antibiotics. The glucose concentration in milk samples was followed by the system enabling the elimination of the effects of bacterial respiration in the output with reliable results in less than 1 min.

Lectin binding studies on a glycopolymer brush flow-through biosensor by localized surface plasmon resonance.

A localized surface plasmon resonance biosensor in a flow-through configuration was applied for investigating kinetics of lectin binding to surface-grafted glycopolymer brushes. Polycarbonate filter membranes with pore sizes of 400nm were coated with a 114-nm thick gold layer and used as substrate for surface-initiated atom-transfer radical polymerization of a glycomonomer. These grafted from glycopolymer brushes were further modified with two subsequent enzymatic reactions on the surface to yield an immobilized trisaccharide presenting brush. Specific binding of lectins including Clostridium difficile toxin A receptor domain to the glycopolymer brush surface could be investigated in a microfluidic setup with flow-through of the analytes and transmission surface plasmon resonance spectroscopy. Graphical abstract Glycopolymer brushes serve as high affinity ligands for lectin and toxin interactions in a sensitive, disposable flow-through LSPR biosensor.

Biosolids Samples processing for Analyses of Pathogens

In this study, a two-step approach was used to develop new sample detection and processing methods that facilitate the application of molecular techniques to uses and disposal of biosolids. In the first phase, ten methods were compared for recovering pathogens in biosolids, and the three top performing methods were selected for optimization and expanded comparison for bacteria, viruses, and helminths. The selected methods were optimized for solid-to-eluent ratio, centrifugation rate, and centrifugation time. Beef-extract based methods resulted in average recoveries ranging from 42–92% and 37–85% for E. coli and Salmonella, respectively. The range of recoveries by the glycine based method were 53–61% and 64–92% for E. coli and Salmonella, respectively. Both of these methods proved superior than the EPA methods which resulted in 29–41% and 26–40% average recoveries for E. coli and Salmonella, respectively. In the second phase, five commercially available kits were compared for removing PCR inhibitiors from sample concentrates. The Qiagen DNA purification kit was selected for integration into the new sample processing protocol which included a phenol-chloroform extraction step upstream of Qiagen cleaning. The lower limit of detection for DNA by the new method was established at between 10 and 100 nanograms in undiluted biosolid sample concentrates. The sample concentrates cleaned by our newly developed method were shown to be compatible with PCR, Real-time PCR, and a flow-through SPR biosensor technique. The new method was also successfully field tested for detecting human adenoviruses in land applied biosolids. This newly developed technique increases the applicability of PCR amplification for the detection of pathogens in biosolids. Utilization of our molecular method also offers a better classification tool for microbial pathogens (Class A and Class B) in biosolids. This title belongs to WERF Research Report Series ISBN: 9781843397779 (eBook)

Chemiluminescence flow biosensor for glucose using Mg-Al carbonate layered double hydroxides as catalysts and buffer solutions

In this work, serving as supports in immobilizing luminol reagent, catalysts of luminol chemiluminescence (CL), and buffer solutions for the CL reaction, Mg-Al-CO3 layered double hydroxides (LDHs) were found to trigger luminol CL in weak acid solutions (pH 5.8). The silica sol–gel with glucose oxidase and horseradish peroxidase was immobilized in the first half of the inside surface of a clear quartz tube, and luminol-hybrid Mg-Al-CO3 LDHs were packed in the second half. Therefore, a novel CL flow-through biosensor for glucose was constructed in weak acid solutions. The CL intensity was linear with glucose concentration in the range of 0.005–1.0mM, and the detection limit for glucose (S/N=3) was 0.1μM. The proposed biosensor exhibited excellent stability, high reproducibility and high selectivity for the determination of glucose and has been successfully applied to determine glucose in human plasma samples with satisfactory results. The success of this work has broken the bottleneck of the pH incompatibility between luminol CL and enzyme activity.

Online Monitoring of Water Toxicity by Use of Bioluminescent Reporter Bacterial Biochips

We describe a flow-through biosensor for online continuous water toxicity monitoring. At the heart of the device are disposable modular biochips incorporating agar-immobilized bioluminescent recombinant reporter bacteria, the responses of which are probed by single-photon avalanche diode detectors. To demonstrate the biosensor capabilities, we equipped it with biochips harboring both inducible and constitutive reporter strains and exposed it to a continuous water flow for up to 10 days. During these periods we challenged the biosensor with 2-h pulses of water spiked with model compounds representing different classes of potential water pollutants, as well as with a sample of industrial wastewater. The biosensor reporter panel detected all simulated contamination events within 0.5-2.5 h, and its response was indicative of the nature of the contaminating chemicals. We believe that a biosensor of the proposed design can be integrated into future water safety and security networks, as part of an early warning system against accidental or intentional water pollution by toxic chemicals.

Development of a novel enzyme reactor and application as a chemiluminescence flow-through biosensor

A novel enzyme reactor was prepared using calcium alginate fiber (CAF) and amine-modified nanosized mesoporous silica (AMNMS) as a support. Combination of the adsorption of the enzyme on AMNMS with the cage effect of the polymer greatly increases the catalytic activity and the stability of the immobilized enzyme. It was shown that the lifetime, stability, and catalytic activity of the enzyme reactor were greatly improved by incorporating AMNMS into CAF to efficiently encapsulate the enzyme. Glucose oxidase was chosen as a model enzyme to explore the possibility of using CAF-AMNMS as a matrix for enzyme immobilization in the design of a chemiluminescence (CL) flow-through biosensor. The sensitivity of the flow-through biosensor combined with a novel luminol-diperiodatonickelate CL system was higher than for other reported CL biosensors. The proposed biosensor exhibits short response time, easy operation, long lifetime, high catalytic activity, high sensitivity, and simple assembly.

Application of a novel co-enzyme reactor in chemiluminescence flow-through biosensor for determination of lactose

A novel enzyme reactor with co-immobilization of β-galactosidase and glucose oxidase in calcium alginate fiber (CAF) and amine modified nanosized mesoporous silica (AMNMS) was prepared which incorporate the adsorption and catalysis of AMNMS with the cage effect of the polymer to increase catalytic activity and stability of immobilized enzyme. The enzyme reactor was applied to prepare a chemiluminescence (CL) flow-through biosensor for determination of lactose combined with a novel luminol-diperiodatonickelate (DPN) CL system we reported. It shows that the CL flow-through biosensor possesses long lifetime, high stability, high catalytic activity and sensitivity. The relative CL intensity was linear with the lactose concentration in the range of 8 × 10 −8–4 × 10 −6 g mL −1 with the detection limit of 2.7 × 10 −8 g mL −1 (3 σ). It has been successfully applied to the determination of lactose in milk.

Biosensor for determination of carboxylic acids in wines based on the inhibition of sarcosine oxidase

The flow-through amperometric biosensor is presented for determination of carboxylic acids. It is based on two sensor layers that are deposited on a platinum electrode. The inner layer serves to eliminate interferences by limiting diffusion of electrochemically active substances such as ascorbic acid. This layer is electro-polymerized using an equimolar mixture of o-phenylenediamine and resorcinol. The outer layer is prepared by cross-linking the enzyme sarcosine oxidase and bovine serum albumin using glutaraldehyde. The formation of enzymatically produced hydrogen peroxide is monitored at 600 mV vs. an Ag/AgCl reference electrode. The addition of carboxylic acids causes competitive inhibition of the enzyme and a decrease in signal. The assay was optimized for determination of carboxylic acids in wine samples. Following 10-fold dilution, most samples contain 1–10 mM individual carboxylic acids and thus a 5 mM concentration of sarcosine was chosen as being optimal for competition. In case of real samples, the biosensor measures the sum of all carboxylic acids, which serves as a parameter describing the quality of wines. Results from testing several wine samples are reported.

Chemiluminescence flow-through biosensor for glucose with eggshell membrane as enzyme immobilization platform

In this study, a new chemiluminescence (CL) flow-through biosensor for glucose was developed by immobilizing glucose oxidase (GOD) and horseradish peroxidase (HRP) on the eggshell membrane with glutaraldehyde as a cross-linker. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and hydrogen peroxide (H 2O 2) and then H 2O 2 oxidizing luminol to produce CL emission in the presence of HRP. The immobilization condition (e.g., immobilization time, GOD/HRP ratio, glutaraldehyde concentration) was studied in detail. It showed good storage stability at 4 °C over a 5-month period. The proposed biosensor exhibited short response time, high sensitivity, easy operation, and simple sensor assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.