1.5 Proteinanalytik
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Type-I pyrethroids are frequently used for disinfection purposes on airplanes from and to tropical areas. The WHO (Word Health Organization) defines “disinfection” as the procedure whereby health measures are taken to control or kill the insect vectors of human diseases including dengue, yellow fever and malaria.1 Due to this, a new effective and rapid method for pyrethroids analysis directly in the plane is needed.
To detect type-I pyrethroids, gated materials are constructed for the purpose of finely tuning the movement of a cargo from voids of a mesoporous inorganic support to a solution, in response to a predefined stimulus.2 Antibodies are bound to specific functional groups grafted on the support’s surface with the two-fold aim of closing the mesopores and working as biological receptors for the target compounds.2 A specific opening mechanism, activated selectively in presence of pyrethroids as analytes, is able to control the release of an indicator dye previously loaded inside the inorganic support. As only few molecules of pyrethroids are necessary to open a pore and release a large amount of dyes, the system shows an intrinsic signal amplification quantified as a fluorescence emission intensity.3
Different mesoporous silica supports (from nano- and microparticles to platelets and short fibers) were synthetized, characterized and functionalized following different capping strategies. In this contribution, we will compare the temporal response behaviour of the optimized gated materials to verify if the respective delivery systems are properly closed and how fast they can be opened in presence of the analytes. Our results indicate that the trends of the kinetics observed in solution show a better control of the closing/opening mechanism when the epitope region of the antibody (around 10 nm) properly fits the pore size of the carriers.
Because the incorporation of an antibody-gated dye-delivery system with a conventional test-strip-based lateral-flow assay allows for the detection of analytes down to the ppb level in an easy-to-operate manner and an overall assay time of 2–5 min, which is fast for a biochemical test,3 we carried out first model studies for a lateral flow test assay on membranes, using a smartphone setup for read-out.
Vor dem Hintergrund der einzigartigen Modularität, hohen Empfindlichkeit und Selektivität von Antikörper‐gesteuerten Indikatorfreisetzungssystemen (gAID‐Systemen) wurde hier ein Multiplex‐Assay für drei organische Explosivstoffmoleküle (TATP, TNT, PETN) entwickelt, der es erlaubt, die Analyten gleichzeitig in flüssiger Phase mit einem einzelnen Teststreifen und einem Fluoreszenzlesegerät bzw. Smartphone als Detektor in Konzentrationen bis in den unteren ppb‐Bereich in <5 min nachzuweisen. Alle drei Systeme, darunter die hier neu entwickelten Systeme für TNT und PETN, tolerieren zudem nicht nur gepufferte wässrige Modelllösungen, sondern auch komplexere Matrices. Neben einem konventionellen Teststreifen mit einem Kanal erlaubte uns die Anwendung von Wachsdrucktechnologie das Herstellen von mehrkanaligen Streifen mit vergleichbarer analytischer Leistungsfähigkeit, was das enorme Potenzial der modular aufgebauten, hybriden Biosensormaterialien im Hinblick auf eine für den Endanwender maßgeschneiderte Vor‐Ort‐Analytik unterstreicht.
The urgent necessity to carry out reliable and relevant analytical measurements directly at a point-of-need is one of the current drivers for the development of miniaturised analytical systems, quick tests and wearables. Despite their simplicity, this type of tests must guarantee analytical relevance and reliability like laboratory-based analysis, e.g., in terms of sensitivity, selectivity, immunity against false positives and false negatives as well as robustness and repeatability. Keeping in mind the high sensitivity offered by gated indicator-releasing micro- and nanoparticles due to their inherent features of signal amplification, we performed several optimisations to develop a potential biosensor platform for use in rapid tests. Conceptually, these gated materials are closely related to drug delivery systems, consisting of high porous materials usually closed with macromolecular “caps” and loaded with indicator molecules that are released in presence of a target analyte. However, the key difference between the two types of functional materials is that many drug delivery systems should deliver their cargo over a longer period, often many hours, whereas the gated materials prepared for sensing should show fast release kinetics, on the order of <5 min.
With the aim to optimise and adapt gated materials for sensing purposes, we prepared in this work several antibody-gated materials for small-molecule sensing. The materials consisted of porous silica particles containing indicator molecules in the pores and certain hapten molecules grafted to the particle surface close to the pore openings. The pores were then capped with antibodies binding to these haptens, thus inhibiting the escape of the indicators from inside of the pores. In presence of the corresponding analyte, the antibody is displaced from the surface of the material, allowing the escape of the indicators. This allows the detection of the analyte indirectly through an inherent signal amplification. In this work, the insecticide permethrin, a type-I pyrethroid, was selected as target model, because type-I pyrethroids play an important role in airplane disinfection. A first in-depth study of the various chemical tuning options of such antibody gated systems was performed. Different mesoporous silica supports, different functionalisation routes and different loading sequences were assessed. The materials’ performances were evaluated by studying their temporal response behaviour and detection sensitivity, including the tightness of pore closure (through the amount of blank release in absence of analyte) and the release kinetics. Our results indicate that the better the paratope-accommodating Fab region of the antibody “cap” fits into the host material’s pore openings, the better the closing/opening mechanism can be controlled. Because such materials can be used in various different formats from suspension assays[1] via microfluidic chips[2] to test strip-based lateral flow assays,[3] such materials present a powerful analytical particle platform for the sensitive analytics and diagnostics outside of a laboratory, realising sensitivities down to the µg kg–1 range in less analysis times of less than 5 min as we have recently demonstrated.[4]
The urgent necessity to carry out reliable and relevant analytical measurements directly at a point-of-need is one of the current drivers for the development of miniaturised analytical systems, quick tests and wearables. Despite their simplicity, this type of tests must guarantee analytical relevance and reliability like laboratory-based analysis, e.g., in terms of sensitivity, selectivity, immunity against false positives and false negatives as well as robustness and repeatability. Keeping in mind the high sensitivity offered by gated indicator-releasing micro- and nanoparticles due to their inherent features of signal amplification, we performed several optimisations to develop a potential biosensor platform for use in rapid tests. Conceptually, these gated materials are closely related to drug delivery systems, consisting of high porous materials usually closed with macromolecular “caps” and loaded with indicator molecules that are released in presence of a target analyte. However, the key difference between the two types of functional materials is that many drug delivery systems should deliver their cargo over a longer period, often many hours, whereas the gated materials prepared for sensing should show fast release kinetics, on the order of <5 min.
With the aim to optimise and adapt gated materials for sensing purposes, we prepared in this work several antibody-gated materials for small-molecule sensing. The materials consisted of porous silica particles containing indicator molecules in the pores and certain hapten molecules grafted to the particle surface close to the pore openings. The pores were then capped with antibodies binding to these haptens, thus inhibiting the escape of the indicators from inside of the pores. In presence of the corresponding analyte, the antibody is displaced from the surface of the material, allowing the escape of the indicators. This allows the detection of the analyte indirectly through an inherent signal amplification. In this work, the insecticide permethrin, a type-I pyrethroid, was selected as target model, because type-I pyrethroids play an important role in airplane disinfection. A first in-depth study of the various chemical tuning options of such antibody gated systems was performed. Different mesoporous silica supports, different functionalisation routes and different loading sequences were assessed. The materials’ performances were evaluated by studying their temporal response behaviour and detection sensitivity, including the tightness of pore closure (through the amount of blank release in absence of analyte) and the release kinetics. Our results indicate that the better the paratope-accommodating Fab region of the antibody “cap” fits into the host material’s pore openings, the better the closing/opening mechanism can be controlled. Because such materials can be used in various different formats from suspension assays[1] via microfluidic chips[2] to test strip-based lateral flow assays,[3] such materials present a powerful analytical particle platform for the sensitive analytics and diagnostics outside of a laboratory, realising sensitivities down to the µg kg–1 range in less analysis times of less than 5 min as we have recently demonstrated.[4]
Methods for the rapid and sensitive detection of target analytes are gaining importance in medical diagnostics and environmental monitoring, in the security, occupational health and safety as well as food sectors. Among all of the methods employed for rapid tests, lateral flow assays (LFAs) are the most commonly used, and hundreds of test kits based on this technique are available on the market. A major drawback is that most of these capture agents either indicate the analyte only indirectly, and in most cases a second binding agent able to bind directly or indirectly to the analytes is necessary (e.g., a secondary labeled antibody). Furthermore, in certain cases in which the (ultra)trace detection of an analyte is required, the traditional approach of a certain number of probe molecules being conjugated to a particular support is not sufficient. Therefore, novel concepts implementing steps of effective signal amplification are urgently required.
Keeping in mind these limitations, we thought that the sensitivity of these systems should be improvable through employment of gated reporter molecule-releasing hybrid nanoparticle materials on novel lateral flow devices. On one hand, the gated sensor material can produce a massive signal amplification, by releasing many reporter molecules only after chemical recognition of a few analyte molecules has taken place in an independent and separate step at the pore openings. On the other hand, the employment of tailored capture materials for the selective interaction with the released reporter molecules in a second arbitrary zone on the strip allows to concentrate or focus the latter for more efficient detection or to create selective multi-spot detection zones, which renders the simultaneous detection of several reporter molecules at the same time in multiplexed detection of various analytes possible.
For that purpose, we have prepared several stimuli-responsive materials for small-molecule sensing based on specific interactions between biomolecules such as antibodies with the corresponding analytes for the detection of certain explosives. In order to prepare these bio-capped materials, we have selected silica mesoporous nanoparticles (MSNs) as inorganic support due to their unique properties such as defined void structure, high inner surface area and flexible functionalization chemistry. These MSNs are loaded with a brightly fluorescent indicator dye, and the external surface is subsequently functionalized with suitable molecules able to interact with antibodies, efficiently inhibiting dye release. The opening protocol and delivery of the entrapped dye is reminiscent of a displacement reaction involving the presence of the target analyte, producing a displacement of the biomolecule and allowing the detection of the target analyte.
The presentation discusses general aspects of system design as well as analytical performance and highlights the integration into a lateral-flow assay, showing as an example the determination of the explosives TATP, TNT and PETN with fluorescence readout, in single-substance and multiplexing modes.