Dorota Wencel

Silica nanoparticles for cell imaging and intracellular sensing

There is increasing interest in the use of nanoparticles (NPs) for biomedical applications. In particular, nanobiophotonic approaches using fluorescence offers the potential of high sensitivity and selectivity in applications such as cell imaging and intracellular sensing. In this review, we focus primarily on the use of fluorescent silica NPs for these applications and, in so doing, aim to enhance and complement the key recent review articles on these topics. We summarize the main synthetic approaches, namely the Stöber and microemulsion processes, and, in this context, we deal with issues in relation to both covalent and physical incorporation of different types of dyes in the particles. The important issue of NP functionalization for conjugation to biomolecules is discussed and strategies published in the recent literature are highlighted and evaluated. We cite recent examples of the use of fluorescent silica NPs for cell imaging in the areas of cancer, stem cell and infectious disease research, and we review the current literature on the use of silica NPs for intracellular sensing of oxygen, pH and ionic species. We include a short final section which seeks to identify the main challenges and obstacles in relation to the potential widespread use of these particles for in vivo diagnostics and therapeutics.

Fabrication and performance evaluation of highly sensitive hybrid sol-gel-derived oxygen sensor films based on a fluorinated precursor.

This paper describes the fabrication and performance of a range of highly sensitive luminescence-based oxygen sensor films based on the fluorinated sol-gel precursor 3,3,3-trifluoropropyltrimethoxysilane (TFP-TMOS). The oxygen-sensitive ruthenium complex [Ruthenium(II)-tris(4,7-diphenyl-1,10-phenanthroline)] dichloride, [Ru(dpp)(3)](2+) was entrapped in a wide range of ORMOSILs (organically modified silicates) matrices composed of alkyl and TFP-TMOS sol-gel precursors in different relative ratios. The influence of TFP-TMOS on sensor sensitivity, humidity-sensitivity and long-term stability was investigated and performance was compared to that of similar but non-fluorinated films. The optimum limit of detection was found to be 0.002% of oxygen for the propyltriethoxysilane (PTEOS)/TFP-TMOS-derived film compared to 0.09% for PTEOS-derived films reported previously. Photobleaching of the luminescent complex in fluorinated and non-fluorinated matrices was also investigated. It was established that photobleaching was reduced but not eliminated in fluorinated films. All films produced in this study exhibit very good reproducibility, reversibility, enhanced sensitivity, humidity-insensitivity and long-term stability.

The development and characterisation of novel hybrid sol–gel-derived films for optical pH sensing

This paper focuses on the development and detailed characterisation of a novel hybrid sol–gel material derived from 3-glycidoxypropyltrimethoxysilane (GPTMS) and ethyltriethoxysilane (ETEOS). The material has been doped with a fluorescent pH-sensitive dye, modified 8-hydroxy-1,3,6-pyrene trisulfonic acid (HPTS) and its application as a high-performance optical ratiometric pH sensor has been demonstrated. The optimum sol–gel-based material composition has been characterised using atomic force microscopy (AFM), scanning electron microscopy in combination with energy dispersive X-ray spectroscopy (SEM-EDX), thermal gravimetric-infrared (TGA-IR) analysis, Fourier transform infrared (FT-IR) and Raman spectroscopy, solid state 29Si and 13C nuclear magnetic resonance (NMR). AFM and SEM images demonstrated a high degree of homogeneity of the pH films. FT-IR, Raman and NMR results have shown that hydrolysis and condensation reactions were almost completed and extensive crosslinking occurred in the final material. They also revealed that an opening of the epoxy ring in GPTMS took place which was key to optimum pH response. The pH sensor produced using the hybrid material compares very well with the current state-of-the-art and exhibits very good reversibility, reproducibility, stability, short response time and no leaching. The dynamic range extends from pH 5.0 to 8.0 which is compatible with bioprocessing, environmental and clinical monitoring applications.

Development of a compact optical sensor for real-time, breath-by-breath detection of oxygen

The detection of oxygen in breath is of central importance to investigations of metabolism and respiration in both clinical and athletic performance monitoring applications. This paper reports the development of a portable, lightweight optical oxygen sensor that is intended to provide a breath oxygen monitoring solution that is deployable outside a laboratory environment. The sensing methodology is based on the detection of changes in the fluorescence emission of an oxygen-sensitive fluorescent dye. The novelty of the system stems from the humidity-insensitive nature of the oxygen sensor, the highly efficient and compact optical configuration and the use of a novel, wearable control unit based on DSP circuitry. These components combine to provide a portable breath oxygen monitor that can detect changes in the in-breath O(2) concentration profile in real-time over a broad range of breathing rates in situations of both rest and exercise. The reported system is expected to have a significant impact on point-of-care (POC) breath-based diagnostics and high performance athletic monitoring.

Breath-by-breath measurement of oxygen using a compact optical sensor

We report on the development of a novel optical oxygen sensor for breath monitoring applications using the technique of phase fluorometry. The principal design criteria are that the system be compact, lightweight, and employ a disposable sensing element (while performing competitively with current commercial analyzers). The oxygen-sensitive, luminescent ruthenium complex Ru[dpp](3)(2+) is encapsulated in a sol-gel matrix and deposited onto a custom-designed, polymer sensor chip that provides significantly improved luminescence capture efficiency. The performance of the sensor module is characterized using a commercially available lung simulator. A resolution of 0.03% O(2) is achieved, which compares well with commercial breath monitoring systems and, when combined with its immunity to humidity and ability to respond effectively across a broad range of breathing rates, makes this device an extremely promising candidate for the development of a practical, low-cost biodiagnostic tool.

Controlled deposition of sol–gel sensor material using hemiwicking

Optical sensors are fabricated by depositing liquid sol–gel sensor material on a polycarbonate surface, which has been decorated with arrays of periodic micropillars. Using the principle of hemiwicking, the liquid material is spread, guided by the surface structures, to homogeneously fill the volume between the surface structures and form a liquid film with a thickness determined by the height of the micropillars. After evaporation of solvents, a uniform layer of sensor material resides on the surface. This fabrication method enables easy and reproducible deposits of isolated spots of different sensor materials of precise thickness to be made on plastic surfaces, and it provides an improved method for fabricating cheap optical sensors integrated in disposable lab containers.

Development of organically modified silica nanoparticles for monitoring the intracellular level of oxygen using a frequency-domain FLIM platform

Monitoring cellular homeostasis is one of the crucial elements in understanding the real causes of the pathological state and in designing more efficient treatments. Fluorescent nanometer sized particles have great potential in the quantitative real-time analysis of important cellular analytes. In this paper we focus on the development of optical chemical nanosensors for probing dissolved oxygen inside living cells. The nanosensor is composed of organically modified silica nanoparticles, doped with a luminescent oxygen-sensitive [Ru(II)-tris(4,7-diphenyl-1,10-phenanthroline)] ([Ru(dpp)3]2+) complex. A monodisperse population of nanoparticles with an average size of 70 nm was obtained based on a modified sol–gel-based Stober method. The nanoparticles were initially calibrated in water using a phase fluorometry setup. Very good repeatability from cycle to cycle and reversibility in oxygen response was obtained for the nanoparticles. The excellent performance of the nanosensors is reflected in their very low limit of detection (LOD) (0.007 ppm). Transmission electron microscopy images obtained from in vitro studies reveals that the final intracellular location of the nanoparticles is in the lysosomes. The performance of the nanoparticles was tested inside cells using Fluorescence Lifetime Imaging Microscopy (FLIM) instrumentation. Despite a decrease in the oxygen sensitivity of the nanoparticles located in the intracellular milieu (LOD = 0.226 ppm), a significant change in lifetime (∼1.3 μs) is detected within the physiological oxygen concentration range. Moreover, nanoparticles show no cytotoxic effect when incubated with cells for up to 72 h. These results demonstrate therefore the great potential of ([Ru(dpp)3]2+)-doped organically modified silica nanoparticles for monitoring the intracellular oxygen concentration.

Synthesis, tailoring and characterization of silica nanoparticles containing a highly stable ruthenium complex

This paper describes the synthesis and characterization of sol-gel silica nanoparticles (NPs) derived from tetraethoxysilane (TEOS) and from tetraethoxysilane and methyltriethoxysilane (TEOS-MTEOS) in which is encapsulated, an in-house synthesized, stable oxygen-sensitive ruthenium complex, ruthenium (II) (bis-2,2-bipyridyl)-2(4-carboxylphenyl) imidazo[4,5-f][1,10]phenanthroline. These NPs were characterized using dynamic light scattering, transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy and Brunauer-Emmett-Teller analysis. The spherical, stable and monodispersed NPs have been prepared using the Stöber method. It was found that the addition of prehydrolyzed MTEOS-based sol prepared in an acidic environment to the reaction mixture containing TEOS NPs synthesized for 6 h produced material with increased porosity when compared to pure silica NPs. Oxygen sensitivity, stability, photobleaching and leaching have been characterized. The hybrid NPs exhibit enhanced O₂ sensitivity but a high degree of leaching when compared to pure silica NPs, which have minimum O₂ sensitivity and no leaching.

Novel hybrid sol-gel materials for smart sensor windows

Current sensor trends, such as multianalyte capability, miniaturisation and patternability are important drivers for materials requirements in optical chemical sensors. In particular, issues such as enhanced sensitivity and printablity are key in developing optimised sensor materials for smart windows for bioprocessing applications. This study focuses on combining novel sol-gel-based hybrid matrices with engineered luminescent complexes to produce stable luminescence-based optical sensors with enhanced sensitivity for a range of analytes including oxygen, pH and carbon dioxide. As well as optimising sensor performance, issues such as surface modification of the plastic substrate and compatibility with different deposition techniques were addressed. Hybrid sol-gel matrices were developed using a range of precursors including tetraethoxysilane (TEOS), methyltriethoxysilane (MTEOS), ethyltriethoxysilane (ETEOS), n-propyltriethoxysilane (PTEOS), phenyltriethoxysilane (PhTEOS), and n-octyltriethoxysilane (C8TEOS). Oxygen sensing, based on luminescence quenching of ruthenium phenanthroline complexes, has been realised with each of these hybrid materials. Furthermore, the possibility of immobilising pH-indicators for pH and carbon dioxide sensing has been investigated with some success. In the context of in-situ monitoring of bioprocesses, issues such as humidity interference as well as the chemical robustness of the multianalyte platform, were addressed.

Dual excitation fluorescence-based sensors for pH and dissolved carbon dioxide monitoring

We report on high performance ratiometric fluorescence-based pH and dissolved carbon dioxide (dCO2) sensors for use in bioprocess and environmental monitoring applications, respectively. Novel hybrid sol-gel-based sensor materials have been developed and successfully used as host matrices in pH and dCO2 sensing. These sensors are easy to prepare and miniaturise, low-cost in terms of fabrication, are mass-producible and when the combination of key performance parameters is considered, compare very well to similar sensor systems which have been reported in the literature in terms of resolution, stability and leaching characteristics. Both pH and dCO2 sensor films can be interrogated with a robust, ratiometric optical probe that combines effective fluorescence excitation and detection and thus facilitates the production of a highly-sensitive sensor system using low-cost optoelectronic components. In the probe configuration both LEDs are operated simultaneously, which represents a significant improvement over existing ratiometric systems where the light sources must by operated sequentially.