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Curved substrates can be micro-structured by laser ablation, which is not possible with standard lithographic methods. The novel femtosecond-pulse laser technique allows the production of defined and reproducible micro-perforations of originally analyte-impermeable membranes. The trans-membrane analyte flux can be controlled both by the variation of the laser focus diameter resulting in different areas of single perforations, and the number of perforations in arrays on small membrane areas. This leads to a higher degree of variability as well as reproducibility of the diffusion qualities of sensor membranes, and marks the main innovation with this technique compared to the hand-made mechanical perforation by specially grinded needles used up to now. Touchless micro-perforation of small membrane areas with negligible heat damage of the structures adjacent to the perforation allows the application of analyte door membranes directly onto curved surfaces of miniaturized needle-sensors assigned for in vivo glucose monitoring, for the first time.
Cardiac troponin I (cTnI) is a crucial biomarker for diagnosing cardiac vascular diseases, including acute myocardial infarction (AMI). This study presents a proof-of-concept chemiluminescence-based immunosensor for rapid and accurate measurement of cTnI, with the potential for online monitoring. The immunosensor incorporates a flow cell design and a sensitive complementary metal-oxide-semiconductor (CMOS) camera for optical readout. A microfluidic setup was established to enable selective and quasi-online determination of cTnI within ten minutes. The sensor was tested with recombinant cTnI in phosphate buffer, demonstrating measurements in the concentration range of 2–25 µg/L, with a limit of detection (LoD) of 0.6 µg/L (23 pmol/L) achieved using the optimized system. The immunosensor exhibited high selectivity, as no cross-reactivity was observed with other recombinant proteins such as cTnT and cTnC at a concentration of 16 µg/L. Measurements with diluted blood plasma and serum yielded an LoD of 60 µg/L (2.4 nmol/L) and 70 µg/L (2.9 nmol/L), respectively. This biosensor offers a promising approach for the rapid and sensitive detection of cTnI, contributing to the diagnosis and management of acute myocardial infarction and other cardiac vascular diseases.
The objective of this study was to introduce differential sensing techniques to synthetic systems that act, like olfactory receptors, as transporters in lipid bilayer membranes. Routine with most alternative chemosensing ensembles, pattern generation has, quite ironically, remained inaccessible in lipid bilayers because the number of available crossresponsive sensor components has been insufficient. To address this challenge, we here report on the use of cationic hydrazides that can react in situ with hydrophobic analytes to produce cationic amphiphiles which in turn can act as countercation activators for polyanionic transporters in fluorogenic vesicles. To expand the dimension of signals generated by this system, a small collection of small peptides containing a positive charge (guanidinium, ammonium) and one to three reactive hydrazides are prepared. Odorants are used as examples for hydrophobic analytes, perfumes to probe compatibility with complex matrices, and counterion-activated calf-thymus DNA as representative polyion–counterion transport system. Principal component and hierarchical cluster analysis of the obtained multidimensional patterns are shown to differentiate at least 21 analytes in a single score plot, discriminating also closely related structures such as enantiomers, cis–trans isomers, single-atom homologs, as well as all tested perfumes. Inverse detection provides access to analytes as small as acetone. The general nature of the introduced methodology promises to find diverse applications in current topics in biomembrane research.
A new type of biodetector was designed based on a bioluminescence test with the bacterium Vibrio fischeri performed in a liquid continuous flow-through system. Here we describe the modification of a commercial tube luminescence detector to work in the flow mode by building a new flow cell holder and a new case including top cover to connect the flow cell with the waste and the incubation capillary in a light-proof manner. As different samples were injected successively it was necessary to keep the individual peaks separated. This was done using an air-segmented flow in the reaction coil. To afford fast screening, the incubation time of the sample and the Vibrio fischeri, which equaled the dead time of the detection system, was set at 5.6 min. Rapid monitoring of toxic substances is achieved by using 20 µL of sample and flow-rates of 110150 µL min-1. As a proof-of-principle, we show results for the detection of five selected di-, tri- and tetrachlorophenols at different concentrations varying from 1 to 200 mg L-1. Calculation of inhibition rates and EC50 values were performed and compared with corresponding values from the DIN EN ISO 11348-2 microplate format. Compared with the latter, the inhibition rates obtained with our flow-through biodetector for the compounds tested were generally about twofold lower, but importantly, a much faster detection is possible.
Silicon photonic micro-ring resonators (MRR) developed on the silicon-on-insulator (SOI) platform, owing to their high sensitivity and small footprint, show great potential for many chemical and biological sensing applications such as label-free detection in environmental monitoring, biomedical engineering, and food analysis. In this tutorial,we provide the theoretical background and give design guidelines for SOI-based MRR as well as examples of surface functionalization procedures for label-free detection of molecules. After introducing the advantages and perspectives of MRR, fundamentals of MRR are described in detail, followed by an introduction to the fabrication methods, which are based on a complementary metal-oxide semiconductor (CMOS) technology. Optimization of MRR for chemical and biological sensing is provided, with special emphasis on the optimization of waveguide geometry. At this point, the difference between chemical bulk sensing and label-free surface sensing is explained, and definitions like waveguide sensitivity, ring sensitivity, overall sensitivity as well as the limit of detection (LoD) of MRR are introduced. Further, we show and explain chemical bulk sensing of sodium chloride (NaCl) in water and provide a recipe for label-free surface sensing.
Silicon photonic micro-ring resonators (MRR) developed on the silicon-on-insulator (SOI) platform, owing to their high sensitivity and small footprint, show great potential for many chemical and biological sensing applications such as label-free detection in environmental monitoring, biomedical engineering, and food analysis. In this tutorial, we provide the theoretical background and give design guidelines for SOI-based MRR as well as examples of surface functionalization procedures for label-free detection of molecules.
After introducing the advantages and perspectives of MRR, fundamentals of MRR are described in detail, followed by an introduction to the fabrication methods, which are based on a complementary metal-oxide semiconductor (CMOS) technology. Optimization of MRR for chemical and biological sensing is provided, with special emphasis on the optimization of waveguide geometry. At this point, the difference between chemical bulk sensing and label-free surface sensing is explained, and definitions like waveguide sensitivity, ring sensitivity, overall sensitivity as well as the limit of detection (LoD) of MRR are introduced. Further, we show and explain chemical bulk sensing of sodium chloride (NaCl) in water and provide a recipe for label-free surface sensing.
Biosensors, as defined by Pure and Applied Chemistry, are ‘chemical sensors in which the recognition System utilizes a biochemical mechanism. The biological recognition system translates information from the biochemical domain, usually an analyte concentration, into a chemical or physical output signal with a defined sensitivity’.(1) It is also appointed that chemical or biological sensors contain two basic components connected in series: a chemical or biomolecular recognition System (receptor) and a physicochemical transducer. According to this prerequisite, this overlook is confined to sensor devices that combine a biomolecular recognition element with an optical signal transducer. Homogeneous or intracellular assays using fluorescent molecular probes or nanoparticles are not considered, although they are frequently termed as molecular sensors or nanosensors in the literature.
Fluorescence-based biosensors are generalized as those devices that derive an analytical signal from a photoluminescent (either fluorescence or phosphorescence) emission process. Chemi- or bioluminescent detection systems are only briefly discussed in this review.
Biosensors are used for a wide variety of tasks, including detection of compounds of biomedical, environmental or defense interest; on-line monitoring for process control; quality control of foodstuffs; selective detection of compounds undergoing a chemical separation; and screening of drug compounds. Advantages of such devices include high selectivity, rapid response times, reusability, amenability to remote analysis, and immunity to electrical interferences. The selective nature of complexation between biomolecule and analyte and the small size of sensor devices can be combined with advanced detection techiques such as total internal reflection (TIR) spectroscopy. This results in an ability to measure analytes in complex matrices with unsurpassed sensitivity. Such samples may include highly scattering components such as milk or whole blood,(11) or relatively inaccessible locations such as groundwater wells, or even intracellular environments. The key limitation of such devices mainly centers on the poor stability of biological compounds, which can lead to a substantial drift in instrumental response over time. The so-called Cambridge Definition appoints another characteristic property of sensors. Therein, they are defined as ‘miniaturized devices which can deliver real-time and on-line information on the presence of specific compounds or ions in even complex samples’. Accordingly, a sensor is expected to respond reversibly and continuously. With the exception of some enzymatic sensors, these conditions are not fulfilled in case of most biosensors. Particularly, in devices where immunological reagents or DNA are used as recognition elements, they show a lack of reversibility and operate only as a ‘one-shot’ screen, without the potential for continuous, quantitative analysis. Nevertheless, the designations immunosensors or DNA sensors became accepted for such analytical or diagnostic tools.
Preactivation Crosslinking - An Efficient Method for the Oriented Immobilization of Antibodies
(2019)
Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.
Preactivation crosslinking – An efficient method for the oriented immobilization of antibodies
(2019)
Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.
Immunoanalytical Techniques, i.e., antibody-based analytical methods, have been used for decades in clinical diagnostics. What makes them attractive for other fields of application is their short time-to-result and high sensitivity. Microplate-based assays such as ELISA have been adopted early in environmental and food analysis. Yet, to make immunoassays even faster, more sensitive, robust, and, most desirable, portable, advanced materials, sometimes developed for other purposes, can be profitably used to achieve these goals. Materials can be novel labels, e.g., chemical or particle labels, such as fluorophores or nanoparticles. Carrier particles, such as magnetic or polymer beads, make it possible to adopt the assays to meso- or microfluidic set-ups and encoding them opens the path to multiplex analysis. Specialty electrodes can enable for higher sensitivity in electrochemical detection. All this broadens the scope of application and lowers effort and cost for analysis at the point-of-need.