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Paper-based SERS test strips
(2016)
For the non-destructive chemical analysis of organic compounds, several different methods such as NMR, UV-vis absorption, IR, Raman, or fluorescence spectroscopy are available. However, all available methods have some restrictions such as the necessity of a large sample amount, interferences in the presence of water, or overlapping signals from the analytes or matrix. Surface enhanced Raman scattering (SERS) allows to observe analytes directly without labelling in low concentrations in aqueous solutions and to identify them by their spectral fingerprint. Therefore, in this work we use SERS as a detection method for different analytes in low concentrations in combination with paper-based test strips as SERS substrates and for sample preparation.
We present a spray method for the preparation of SERS test stripes.[1] With this spray method, nanoparticle solution was deposited on cellulose and glass fibre paper as SERS substrate. The prepared paper-based test strips were tested with classical SERS reporter molecules, e.g. rhodamine 6G, 4-aminothiophenol, and adenine. For the quantification of analytes, highly reproducible signal intensities are necessary, which can be realized with the test strips in acceptable quality. Moreover, employing intensity vs concentration calibration for the analytes, data analysis revealed a behaviour that was best described by a Langmuir isotherm, stressing the strong distance dependence of the SERS effect.
For an easier identification of analytes in a mixture of compounds, the paper-based test strips were functionalised with hydrophobic barriers by wax printing. With these microfluidic paper-based analytical devices (µPAD) the sample mixture can be separated by the chromatographic effects of the paper and the different analytes can be separately detected and identified by SERS.
[1] A. Bolz, U. Panne, K. Rurack, M. Buurman, Glass fibre paper-based test strips for sensitive SERS sensing, Anal. Methods, 2016, 8, 1313-1318.
Surface enhanced Raman scattering (SERS) is a fast and sensitive spectroscopic method for the identification of analytes. With available portable Raman spectrometers, on-site analysis is possible. However, for on-site analysis, SERS substrates, which are cheap, easy to prepare, and simple in sample handling are necessary. Relevant analytes in the addressable concentration region for SERS are e.g. antibiotics and pesticides. Here, we present paper-based test strips for SERS analysis which are coated with silver nanoparticles. The coating was realized with different deposition methods of nanoparticle solutions. The papers were also functionalised with hydrophobic barriers to create μPADs. The strips were tested with selected analytes (e.g., adenine, rhodamine-6G) over a broad concentration range. The focus of our study lay on reproducibility and optimum SERS signal intensity.
For the quantification of analytes, highly reproducible signal intensities are necessary. We have realized this reproducibility in acceptable quality. Moreover, employing intensity vs concentration calibration for the analytes, data analysis revealed a behaviour that was best described by a Langmuir isotherm, stressing the strong distance dependence of the SERS effect. For a fast and reproducible analysis of the data, a Labview program was finally compiled, which was fed with the calibration data and derived the concentration of analyte unknowns accordingly.
The fast identification and quantification of analytes in the field of food safety or environmental analysis is difficult. Surface enhanced Raman scattering (SERS) is an analytical method which can be used simultaneously for the rapid identification and concentration determination of trace analytes,[1,2] usually covering a large dynamic range from nanomolar up to molar concentrations. The identification of the molecules is accomplished through the specific fingerprint of a molecule’s Raman spectrum.
For facile and straightforward SERS measurements, we present here a combination of paper-based SERS test strips with microfluidic systems on paper as a microfluidic paper-based analytical device (μPAD). The SERS μPAD is thus principally suited for cheap, fast, non-destructive, label-free and portable detection of analytes. In this system basically, the use of the microfluidic structured paper increases the sensitivity and suppresses background signals of the SERS assay.
Deposition of the SERS substrate on the test strips is simple and relies on an inkjet printer. For the optimization of the reproducibility and intensity of the SERS signal, we tested different nanoparticles, different numbers of print cycles and different paper types. The nanoparticle solutions used in the μPAD preparation were gold and silver nanoparticle solutions. The paper types were cellulose and glass fiber. SERS arrays were prepared by printing and compared to arrays prepared by spraying. The optimized μPAD was used for the identification and quantification of pure analyte solutions (e.g., adenine) and mixtures of compounds, the concentration series following Langmuir isotherms.
Relevant analytes in the field of food safety are antibiotics and pesticides. We apply the SERS microfluidic paper-based analytical devices for the detection of antibiotics (enoxacin, enrofloxacin) and pesticides.
Microfluidic paper-based analytical devices (μPADs) in combination with surface enhanced Raman scattering (SERS) provide a way for analyses of complex mixtures. The μPADs can be used for the chromatographic separation of different compounds of mixtures in combination with the separate detection of the analytes in different zones on the paper by SERS. SERS allows to observe analytes directly without labelling in low concentrations in aqueous solutions and to identify them by their spectral fingerprint.
SERS substrates on the μPADs were created by drying standard silver nanoparticle (AgNP) solution on the paper. The microfluidic structure of the μPADs was prepared by wax printing. As a model system, an aqueous solution of the non-fluorescent analyte adenine and two fluorescent dyes tris(2,2’-bipyridyl)dichlororuthenium(II) and sulforhodamine B was tested. The dependency of the SERS signal intensity on the analyte concentration can be fitted using a Langmuir isotherm curve progression. With this approach, a semi-quantitative analysis of the components is possible. The reproducibility and stability of the measurement procedure was tested with several measurements over time, different NP batches, and with different analytes in different concentrations and resulted in an average relative standard deviation of 16 %.
The SERS spectra of the mixture of the model system are dominated by one compound depending on the concentration ratio. For the detection and identification of all components of the mixture, the compounds were therefore separated on the μPADs and measured at different positions. The position of adenine on the μPADs is dependent on the AgNP coverage of the paper. Due to this effect, it possible to detect adenine on a defined point on the μPADs and to get an information on the concentration in a mixture of three components.
Multifunctional composite nanoprobes consisting of iron oxide nanoparticles linked to silver and gold nanoparticles, Ag–Magnetite and Au–Magnetite, respectively, were introduced by endocytic uptake into cultured fibroblast cells. The cells containing the non-toxic nanoprobes were shown to be displaceable in an external magnetic field and can be manipulated in microfluidic channels. The distribution of the composite nanostructures that are contained in the endosomal system is discussed on the basis of surfaceenhanced Raman scattering (SERS) mapping, quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping, and cryo soft X-ray tomography (cryo soft-XRT). Cryo soft-XRT of intact, vitrified cells reveals that the composite nanoprobes form intra-endosomal aggregates. The nanoprobes provide SERS signals from the biomolecular composition of their surface in the endosomal environment. The SERS data indicate the high stability of the nanoprobes and of their plasmonic properties in the harsh environment of endosomes and lysosomes. The spectra point at the molecular composition at the surface of the Ag–Magnetite and Au–Magnetite nanostructures that is very similar to that of other Composite structures, but different from the composition of pure silver and gold SERS nanoprobes used for intracellular investigations. As shown by the LA-ICP-MS data, the uptake efficiency of the magnetite composites is approximately two to three times higher than that of the pure gold and silver nanoparticles.
Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLS functionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.
Gold nanostructures that serve as probes for nanospectroscopic analysis of eukaryotic cell cultures can be obtained by the in situ reduction of tetrachloroauric acid (HAuCl4). To understand the formation process of such intracellularly grown particles depending on the incubation medium, the reaction was carried out with 3T3 fibroblast cells in three different incubation media, phosphate buffer, Dulbecco's Modified Eagle Medium (DMEM), and standard cell culture medium (DMEM with fetal calf serum). The size, the optical properties, the biomolecular corona, and the localization of the gold nanoparticles formed in situ vary for the different conditions. The combination of surface-enhanced Raman scattering (SERS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) microscopic mapping and transmission electron microscopy (TEM) provides complementary perspectives on plasmonic nanoparticles and non-plasmonic gold compounds inside the cells. While for the incubation with HAuCl4 in PBS, gold particles provide optical signals from the nucleus, the incubation in standard cell culture medium leads to scavenging of the toxic molecules and the formation of spots of high gold concentration in the cytoplasm without formation of SERS-active particles inside the cells. The biomolecular corona of nanoparticles formed in situ after incubation in buffer and DMEM differs, suggesting that different intracellular molecular species serve for reduction and stabilization. Comparison with data obtained from ready-made gold nanoparticles suggests complementary application of in situ and ex situ generated nanostructures for optical probing.
Thin metal-filled polyterafluoroethylene films with various metal concentration were produced by co-deposition in vacuum. Metal nanocluster size increased with metal concentration. Films were heated up to 300 degrees C, their optical spectra were recorded during heating. The changes in plasmon band shape and wavelength of the nanocluster ensemble during heating are not linearly related with metal concentration and heating temperature. This is caused by different thermal behavior of the complex processes, which are taking place in each of the two materials present in the film. The metal cluster size and optical properties of the whole ensemble can be purposefully formed by varying metal nature, its concentration and annealing temperature of the film. Nano- and micro-domains with properties different from original film were generated by focused excimer laser or electron beam. Gold-filled PTFE nano-structured films were used as substrate for surface enhanced Raman scattering measurements of ultrathin film of Rhodamine 6G dye.
The field of epigenetics describes the relationship between genotype and phenotype, by regulating gene expression without changing the canonical base sequence of DNA. It deals with molecular genomic information that is encoded by a rich repertoire of chemical modifications and molecular interactions. This regulation involves DNA, RNA and proteins that are enzymatically tagged with small molecular groups that alter their physical and chemical properties. It is now clear that epigenetic alterations are involved in development and disease, and thus, are the focus of intensive research. The ability to record epigenetic changes and quantify them in rare medical samples is critical for next generation diagnostics. Optical detection offers the ultimate single-molecule sensitivity and the potential for spectral multiplexing. Here we review recent progress in ultrasensitive optical detection of DNA and histone modifications.