Characterization of microtiterplates with integrated optical sensors for oxygen and pH, and their applications to enzyme activity screening, respirometry, and toxicological assays

https://doi.org/10.1016/j.snb.2005.07.056Get rights and content

Abstract

This paper describes properties of microplates (MTPs) with integrated, fluorescence-based sensors for pH and oxygen, and their application for enzyme screening and monitoring of bacterial respiratory activity. Thin, hydrophilic sensing films consisting of an analyte-sensitive indicator and a reference fluorophore, are deposited on the bottom of the MTP. This allows for calibration-free quantification of pH and pO2 with an acceptable accuracy and resolution for applications such as enzyme activity screening, respirometry, or toxicological assays. The sensor properties are mainly investigated with respect to their qualification for such applications. Specifically, enzyme activity screening is demonstrated for glucose oxidase using oxygen-sensitive plates, and for bacterial growth monitoring of Escherichia coli and Pseudomonas putida. Furthermore, a toxicological assay monitoring the respiration activity of P. putida was converted into a microplate format.

Introduction

Microtiterplates (MTPs) are simple and efficient tools in clinical routine diagnostic and high-throughput screening applications. Introducing optical sensor technology into the microplate format paves the way for new applications in the field of enzyme activity screening or detection of the respiratory activity of different types of cells [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. Two different schemes to combine optical sensors and MTP assays are possible. In the first, the indicator is dissolved or suspended in the sample [1], [2], [3], [4], while in the second it is embedded in a polymer matrix, thus forming a thin film at the bottom of the MTP well [5], [6], [7], [8], [9], [10].

Both approaches have specific advantages and suffer from certain limitations. By using dissolved indicators (or dispersed nanospheres with integrated sensors), the detected parameter represents an average over the whole sample volume. Thus, heterogeneities of the analyte concentration, which are often inevitable in MTP assays due to incomplete mixing [11] or gas exchange with the environment, are not taken into account. This can be critical in cases of sensing oxygen because of oxygen diffusion into the sample. As a result, the surface regions of solutions consuming oxygen at a fast rate will be more oxygenated than the sample located on the bottom. Furthermore, a dissolved indicator often involves other disadvantages like cumbersome handling, inaccurate dispensing, or inhibition if not toxicity towards sample components. On the other hand, dissolved indicators warrant fast response and flexibility in the MTP format.

When using an indicator dye immobilized in a polymer, several demands have to be fulfilled: Thin sensor layers are preferable to realize short response times which permit on-line detection of the analyte. An additional polymer foil (acting as sensor support [5]) should be avoided in order to prevent long response times and complication of the process of fabrication. Small amounts of sensor materials and simple fabrication keep the costs low, which is important for high-throughput applications. Internal referencing of the sensor signal can compensate for varying thicknesses of the sensor spot, resulting in good reproducibility of the signal. Optimally, calibration-free sensors can be used.

Here, MTPs equipped with opto-chemical sensors for pH and oxygen are described which fulfill the above demands. The sensors comprise a fluorescent indicator dye embedded in a polymer together with a reference dye. A solution of the sensor materials in a solvent is placed in the wells of a 96-well MTP using a pipetting robot, generating a thin polymer layer after evaporation of the solvent. The sensors are fully characterized, and enzyme kinetics and respiratory measurements are presented as typical applications. We also demonstrate that pH detection is an advantageous alternative in monitoring respiratory activity compared to approaches using oxygen sensors [1], [2], [3], [5], [7], [8], [9], [10].

Section snippets

Sensing pH

pH-sensitive microtiterplates (MTPs) of type Hydroplate HP96U were obtained from PreSens Precision Sensing (www.presens.de). For characterization of the sensor, either standard buffer solutions (Merck, www.merck.de) or self-made phosphate buffered saline (PBS) solutions of a defined buffer concentration (sodium dihydrogen phosphate and disodium hydrogen phosphate from Merck), ionic strength (IS) and pH were used. The IS was adjusted with sodium chloride (Merck). The pH values of the PBS

Sensor composition

The HydroPlate HP96U makes use of a fluorescein derivative as a pH indicator. It is covalently attached to oxygen-impermeable nanospheres of polyacrylonitrile containing the luminescent ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline complex as a reference dye. This sensing material was dispersed together with carbon black (which acts as optical isolation) in a polyurethane hydrogel matrix. The sensor layer has a final thickness of about 10 μm and is fixed at the bottom of each well of a

Conclusion

We describe the properties and applications of microtiterplates (MTPs) equipped with fluorescent sensors for pH and oxygen. The sensors contain an indicator and an inert reference dye. Referencing warrants accuracy and resolution, and this allows for calibrating only a few sensor spots per lot of MTPs. The dyes are immobilized in a polymer which prevents leaching. Both sensors show fast response times. Their spectral properties allow for the use of standard filters and one-wavelength

Acknowledgement

We thank the Deutsche Bundesstiftung Umwelt (DBU) for financial support (Project 13040/15).

Sarina Arain graduated in chemistry at the University of Regensburg at the Institute of Analytical Chemistry 2001. Since 2001 she is a Ph.D. student at the same Institute. Her research interests are optical sensor technology and the application of optical sensors in biotechnology.

References (15)

There are more references available in the full text version of this article.

Cited by (63)

  • Nanomaterials for Intracellular pH Sensing and Imaging

    2019, Novel Nanomaterials for Biomedical, Environmental and Energy Applications
  • Nanomaterials for Intracellular pH Sensing and Imaging

    2018, Novel Nanomaterials for Biomedical, Environmental and Energy Applications
  • Quantum dots as a possible oxygen sensor

    2014, Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
    Citation Excerpt :

    Particularly noteworthy are the sensors with the use of fluorescence techniques because of their very high sensitivity and selectivity. The essence of this type of sensors is an immobilization in the sensor layer fluorophores whose emission is sensitive to the presence of a determined reagent or to a change in the tested system properties [1–4]. Oxygen is a very important element involved in biological and chemical processes, so there is special group of sensors based on photoluminescence, which enable determination of its concentration [5–7].

View all citing articles on Scopus

Sarina Arain graduated in chemistry at the University of Regensburg at the Institute of Analytical Chemistry 2001. Since 2001 she is a Ph.D. student at the same Institute. Her research interests are optical sensor technology and the application of optical sensors in biotechnology.

Otto S. Wolfbeis is Professor of Analytical Chemistry at the University of Regensburg, Germany. He has authored many papers and reviews on optical (fiber) chemical sensors, fluorescence spectroscopy, and fluorescent probes, and has edited a book on Fiber Optic Chemical Sensors and Biosensors. He acts as the editor of the Springer Series on Chemical Sensors and Biosensors, is the Editor-in-Chief of Microchimica Acta, and acts as the chairman of the steering committees of the biannual conferences on Optical Chemical Sensors and Biosensors (Europt(r)ode). His research interests are in optical chemical sensing and biosensing, in the design of novel schemes in analytical fluorescence spectroscopy, in fluorescent probes, beads, and labels, in biosensors based on thin gold films and molecular imprints, in the design of advanced materials for use in (bio)chemical sensing, and in biomedical and applications of such sensors and analytical schemes.

Ingo Klimant graduated in analytical chemistry at the Mining Acadamy Freiberg (Germany) and received his Ph.D. in chemistry 1993 from the Karl Franzens University of Graz (Austria). After a several years stay at the Max Planck Institute of Marine Microbiology in Bremen (Germany) he was an assistant professor at the University of Regensburg (Germany). Since 2001 he is a full professor at the University of Technology Graz (Austria) and now the head of the Institute of Analytical Chemistry and Radiochemistry. His research interests are sensor technology, spectroscopic methods, material science (in particular micro- and nanostructured materials).

View full text