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Faecal contaminants in water are considered serious threats for human health, due to the presence of viruses, bacteria and other harmful microorganisms.1 Urobilin (UB) is a well-known faecal pigment and can be used as a marker for faecal matter in water.2 UB is commonly present in the urine of all mammals as the catabolic end product of bilirubin degradation.2 As the only simple chemical approach to its detection, Schlesinger’s test is usually used to enhance the weak fluorescence of UB in alcoholic media by complexation with Zinc.2, 3 The major limitation of this method is the only weak enhancement of the intrinsically weak UB fluorescence in aqueous media.3 This work presents an approach to introduce different Zn salts for improved fluorescence response, where we found a clear dependence of the fluorescence yield of UB-Zn(II) complexes on the counterion of the salt in water. By employing a combination of fluorescence parameters like transition energy, fluorescence intensity, and fluorescence lifetime, a photophysical understanding of the structure and conformation of the UB-Zn(II) complexes responsible for the fluorescence enhancement in water could be gained. The possibilities of developing a sensitive analytical method based on the acquired understanding are also discussed.
Glycoproteins are abundant on the cell surface of mammals, providing structural support, modulating cell Membrane properties, and acting as signaling agents. Variation of glycosylation patterns has been found to indicate various disease states, including cell malignancy. Sialic acid (SA) is present as a terminating group on cell-surface glycans, and its overexpression has been linked to several types of cancer. Detection of SA on the cell surface is therefore critical for detection of cancer in its early stages. In this work, a fluorescent molecularly imprinted polymer layer targeting SA was synthesized on the surface of silica-coated polystyrene (PS) particles. Compared to previous works, a PS core supplies a lighter, lower-density support for improved suspension stability and scattering properties. Moreover, their smaller size provides a higher surface-area-to-volume ratio for binding. The incorporation of a fluorescent monomer in the MIP shell allowed for simple and rapid determination of binding specificity in polar media due to a deprotonation−reprotonation interaction mechanism between the fluorescent monomer and SA, which led to spectral changes.
Upon titration of the MIP particles with SA in suspension, an increase in fluorescence emission of the particles was observed, with the MIP particles binding SA more selectively compared to the nonimprinted polymer (NIP) control particles. In cell staining experiments performed by flow cytometry, the binding behavior of the MIP particles compared favorably with that of SA-binding lectins. NIPs prepared with a “dummy” template served as a better negative control in cell binding assays due to the favorable inward orientation of template-binding functional groups in the polymer shell, which reduced nonspecific binding. The results show that fluorescent MIPs targeting SA are a promising tool for in vitro fluorescence staining of cancerous cells and for future diagnosis of cancer at early stages.
Sphingosine-1-phosphate (S1P) is a bioactive sphingo-lipid with a broad range of activities coupled to its role in G-protein coupled receptor signalling. Monitoring of both intra and extra cellular levels of this lipid is challenging due to its low abundance and lack of robust affinity assays or sensors. We here report on fluorescent sensory core-shell molecularly imprinted polymer (MIP) particles responsive to near physiologically relevant levels of S1P and the S1P receptor modulator fingolimod phosphate (FP) in spiked human serum samples. Imprinting was achieved using the tetrabutylammonium (TBA) salt of FP or phosphatidic acid (DPPA·Na) as templates in combination with a polymerizable nitrobenzoxadiazole (NBD)-urea monomer with the dual role of capturing the phospho-anion and signalling its presence. The monomers were grafted from ca 300 nm RAFT-modified silica core particles using ethyleneglycol dimethacrylate (EGDMA) as crosslinker resulting in 10–20 nm thick shells displaying selective fluorescence response to the targeted lipids S1P and DPPA in aqueous buffered media. Potential use of the sensory particles for monitoring S1P in serum was demonstrated on spiked serum samples, proving a linear range of 18–60 μM and a detection limit of 5.6 μM, a value in the same range as the plasma concentration of the biomarker.
The employment of type-I pyrethroids for airplane disinfection in recent years underlines the necessity to develop sensing schemes for the rapid detection of these pesticides directly at the point-of-use. Antibody-gated indicator-releasing materials were thus developed and implemented with test strips for lateral-flow assay-based analysis employing a smartphone for readout. Besides a proper matching of pore sizes and gating macromolecules, the functionalization of both the material's outer surface as well as the strips with PEG chains enhanced system performance. This simple assay allowed for the detection of permethrin as a target molecule at concentrations down to the lower ppb level in less than 5 minutes.
Real-time monitoring of newly acidified organelles during autophagy in living cells is highly desirable for a better understanding of intracellular degradative processes. Herein, we describe a reaction-based boron dipyrromethene (BODIPY) dye containing strongly electron-withdrawing diethyl 2-cyanoacrylate groups at the α-positions. The probe exhibits intense red fluorescence in acidic organelles or the acidified cytosol while negligible fluorescence in other regions of the cell. The underlying mechanism is a nucleophilic reaction at the central meso-carbon of the indacene core, resulting in the loss of π-conjugation entailed by dramatic spectroscopic changes of more than 200 nm between its colorless, non-fluorescent leuco-BODIPY form and its red and brightly emitting form. The reversible transformation between red fluorescent BODIPY and leuco-BODIPY along with negligible cytotoxicity qualifies such dyes for rapid and direct intracellular lysosome imaging and cytosolic acidosis detection simultaneously without any washing step, enabling the real-time monitoring of newly acidified organelles during autophagy.
Chlorination procedures are commonly applied in swimming pool water and wastewater treatment, yet also in food, pharmaceutical, and paper production. The amount of chlorine in water needs to be strictly controlled to ensure efficient killing of pathogens but avoid the induction of negative health effects. Miniaturized microfluidic fluorescence sensors are an appealing approach here when aiming at online or at-site measurements. Two meso-enamine-substituted boron dipyrromethene (BODIPY) dyes were found to exhibit favorable indication properties, their reaction with hypochlorite leading to strong fluorescence enhancement. Real-time assays became possible after integration of these fluorescent probes with designed two-dimensional (2D) and three-dimensional (3D) microfluidic chips, incorporating a passive sinusoidal mixer and a microhydrocyclone, respectively. A comparison of the two microfluidic systems, including their abilities to prevent accumulation or circulation of microbubbles produced by the chemical indication reaction, showed excellent fluidic behavior for the microhydrocyclone-based device. After coupling to a miniaturized optical reader for fluorescence detection, the 2D microfluidic system showed a promising detection range of 0.04−0.5 mg L−1 while still being prone to bubble-induced fluctuations and suffering from considerably low signal gain. The microhydrocyclone-based system was distinctly more robust against gas bubbles, showed a higher signal gain, and allowed us to halve the limit of detection to 0.02 mg L−1. The use of the 3D system to quantify the chlorine content of swimming pool water samples for sensitive and quantitative chlorine monitoring was demonstrated.
Invited for this month’s cover picture is the group of Dr. Knut Rurack at the Department of Analytical Chemistry; Reference Materials at the Bundesanstalt fuer Materialforschung und -pruefung (BAM) in Berlin (Germany). The cover picture shows how differences in color and fluorescence on a test strip can be easily read out with a mobile device. Two reference spots Frame the sensitive spot that indicates the presence of trace amounts of HgII below the threshold in a natural water sample. This dipstick contains a hybrid material that combines boron-dipyrromethene (BODIPY) probes sterically loaded into specifically tailored mesoporous silica particles, allowing for ultrasensitive HgII detection through enhanced fluorescence in a few seconds. The applicability in real water samples and fish extracts are also studied.
The synthesis, characterization, and application of mesoporous materials containing boron–dipyrromethene (BODIPY) moieties that allow the sensitive and selective detection of HgII in aqueous environments by fluorescence enhancement is reported. For this purpose, BODIPY dye I containing a thia‐aza crown ether receptor as the fluorescent probe for the detection of HgII in aqueous environments is encapsulated into mesoporous materials to avoid self‐quenching or aggregation in water. Determination of HgII is accomplished within a few seconds with high selectivity and sensitivity, reaching a limit of detection of 12 ppt. The determination of trace amounts of HgII in natural waters and in fish extracts is demonstrated by using our sensing material. The incorporation of the material into several μ‐PAD strips yields a portable, cheap, quick, and easy‐to‐handle tool for trace HgII analysis in water.
Cyanide is known to be a very hazardous and toxic substance. Through the binding to cytochrome oxidase it inhibits the oxygen utilization in cells. The LD50 of cyanide is as low as 1.0 mg/kg. Therefore it is inalienable to develop sensing methods to detect cyanide with a very high selectivity and sensitivity.
In our group we developed a non-fluorescent monomer which is able to detect cyanide-anions with very high selectivity and sensitivity based on a “turn-on” fluorescence method. As cyanide-source we used tetrabutylammoniumcyanide (TBAC). Using an excitation wavelength of 600 nm the increase in fluorescence at 642 nm is strictly selective with cyanide-anions. Different anions like fluoride, hydroxide or acetate show no increase at all. With this new detection system, concentrations as low as 1.6 nM can be detected.
Molecularly Imprinted Polymers with Integrated Fluorescence as Versatile Biomimetic Sensing Matrices
(2018)
Molecularly imprinted polymers (MIPs) are an established, versatile and high-performance matrix for the selective separation or enrichment of (bio)chemical species, especially small molecules of biochemical or environmental relevance. MIPs are prepared through the polymerization of a mixture of functional monomers and cross-linkers in the presence of the template with subsequent extraction of the latter. Conceptionally, this process can be seen as mimicking in a strongly accelerated, though single-step manner a biological process such as antibody formation. Because the resulting MIPs contain cavities in their matrix that are complementary in size, shape and electronic/ electrostatic or hydrogen bonding demand to the imprinted target molecule or template, these polymers are frequently termed “artificial antibodies”. Compared to natural antibodies, they are chemically and physically much more robust. Regarding sensitivity and selectivity, however, there is still a gap to bridge before MIPs can fully compete with antibodies.
Another favorable aspect that distinguishes MIPs from antibodies is that they can be endowed with an explicit function, allowing the use of MIPs in applications that require more than only an efficient binder. For instance, if specifically designed and polymerizable fluorescent indicators are integrated as functional monomers into a MIP, direct fluorescence sensing can be accomplished. Because MIPs can be prepared in a variety of different formats, their combination with miniaturized or other specific analytical techniques or sensory devices is possible, especially when the transduction mode is light. This presentation will introduce basic design considerations, challenges, limitations and the potential that lies with such sensor materials with some recent examples of our group, targeting various organic oxoanions as analytes.