We present the SERS-based detection of biomolecules in complex biological matrices. This includes the detection of co-factors in bacterial membranes, drugs and its metabolites in clinically relevant matrices as well as cancer-relevant marker in saliva. To overcome the complexity of the biological matrix, a high affinity of the target analyte is essential, which is improved by sample preparation processes or specific enrichment on the sensor surface.
The potential of surface enhanced Raman spectroscopy (SERS) in bioanalytics is illustrated by the example for detecting the antibiotic ciprofloxacin in pharmaceutical formulations in its relevant concentrations. Both, Raman and SERS spectroscopy are applied to identify the target analyte dependent on the complexity of the matrices.
Powerful detection schemes in bioanalytics are associated with the requirements for molecular specificity, high sensitivity and fast detection times. Surface enhanced Raman spectroscopy (SERS) is known to meet those requirements and the strong capability of this method in bioanalytical detection schemes is due to the enhancement of the molecular specific Raman fingerprint by 6 to 8 orders of magnitude. In order to increase the spatial resolution to the nanometer scale, SERS is combined with scanning probe microscopy creating tip enhanced Raman spectroscopy (TERS). Within this presentation, a literature review on SERS and TERS in bioanalytics is given.
In order to allow for a molecular specific as well as trace level sensitive detection in biosensing, vibrational spectroscopy is applied in combination with plasmonic active materials. Within this contribution, we report on the surface-enhanced Raman spectroscopy (SERS) technique, the fabrication of powerful SERS substrates and finally illustrate the potential in biosensing. Mannose-modified SERS tags are applied to be taken up by macrophages in a large amount which serves as a model for the detection of vulnerable atherosclerotic plaques in an early stage. Moreover, to allow for an improved quantification SERS active surfaces are equipped with an internal standard. Finally, a newly developed fabrication strategy of SERS substrate templates will be introduced forming 3D hierarchical nanostructures by metastable state assisted atomic layer deposition. Thus, a detailed overview on innovative plasmonic nanostructures for biosensing developed in our labs is given.
Magnetic nanoparticles (MNPs) have a major role as contrast agent in diagnostic imaging and therapeutic monitoring. In order to research on MNP exposition, degradation and elimination of those nano composites as well as the consequences of the MNP exposition in relation with social economic relevant diseases (cancer, infectious diseases), the comprehensive characterization of magnetic and structural properties is of high importance. Within this contribution, the magnetic characterization of theranostic relevant MNPs is introduced. Applying a vibrating sample magnetometer (VSM), it is found, that the nanocomposites show superparamagnetic behavior and the recorded data confirm iron oxide cores (magnetite/maghemite). Employing Raman spectroscopy, the typical fingerprint information of magnetite is detected. By increasing the laser power, the transition to maghemite and hematite due to the oxidation of the magnetic core is illustrated. Moreover, IR spectroscopy is applied to characterize the coating material e.g. starch or other biocompatible polymers. To determine the stability of MNPs as well as the MNP’s elimination under physiological conditions, different buffer systems were tested i.e. simulated body fluid (SBF) and artificial lysosomal fluid (ALF). The investigated MNPs are stable in SBF; thus, the stability in blood after injection of the contrast agent is guaranteed. Finally, the storage in ALF leads to a complete decomposition of the MNPs, which reflects the conditions in lysosomes and guarantee for a fast MNP elimination.
Acknowledgement: We thank the Federal Ministry of Education and Research (BMBF), Germany as well as the Project Management Jülich (PTJ), Germany for funding the research project NanoBEL (03XP0003F).
Cardiovascular diseases are the leading cause of death worldwide. Atherosclerosis is closely related to the majority of these diseases, as a process of thickening and stiffening of the arterial walls through accumulation of lipids, which is a consequence of aging and life style. Atherosclerosis affects all people in some extent, but not all arterial plaques will necessarily lead to the complications, such as thrombosis, stroke and heart attack. One of the greatest challenges in the risk assessment of atherosclerotic depositions is the detection and recognition of plaques which are unstable and prone to rupture. These vulnerable plaques usually consist of a lipid core that attracts macrophages, a type of white blood cells that are responsible for the degradation of lipids. It has been hypothesized that the amount of macrophages relates to the overall plaque stability. As phagocytes, macrophages also act as recipients for nanoscale particles or structures. Administered gold nanoparticles are usually rabidly taken up by macrophages residing within arterial walls and can therefore be indirectly detected. A very sensitive strategy for probing gold nanoparticles is by utilizing surface enhanced Raman scattering (SERS). By modifying the surface of these particles with SERS active labels it is possible to generate highly specific signals that exhibit sensitivity comparable to fluorescence. SERS labeled gold nanoparticles have been synthesized and the uptake dynamics and efficiency on macrophages in cell cultures was investigated using Raman microscopic imaging. The results clearly show that nanoparticles are taken up by macrophages and support the potential of SERS spectroscopy for the detection of vulnerable plaques.
Acknowledgements: Financial support from the Carl Zeiss Foundation is highly acknowledged. The project “Jenaer Biochip Initiative 2.0” (03IPT513Y) within the framework “InnoProfile Transfer – Unternehmen Region“ is supported by the Federal Ministry of Education and Research, Germany (BMBF).
Infrared spectroscopy enables the label-free detection of structure specific fingerprints of analytes. The sensitivity of corresponding methods can strongly be enhanced by attaching analytes on plasmonic active surfaces.
We introduce a slit array metamaterial perfect absorber (SAMPA) [1] consisting of a dielectric layer sandwiched between two Au layers of which the upper layer is perforated with a periodic array of slits. This structure combines the principle of Extraordinary Optical Transmission (more light is transmitted through a hole than is incident on its surface) with that of Perfect Absorption (reflectance and transmittance are virtually zero). Accordingly, within the slights the electric fields are strongly enhanced and light-matter interaction is correspondingly greatly amplified. Thus, already small concentrations of analytes down to a monolayer can be detected and identified by their spectral fingerprints with a standard mid-infrared spectrometer.
Closely related to the SAMPAs are plasmonic slit absorbers, which simply consist of slit arrays in thin gold layers deposited on a layer of Si3N4.[2] These slit arrays operate like unstructured gold layers if the incident light is polarized parallel to the long slit axes. In contrast, for light polarized perpendicular to the long slit axis, the plasmon is excited. By the introduction of a second slit, which is rotated relative to the first slit, both principal polarization states excite plasmon resonances which can be made to differ in wavelength. As a consequence, the operating wavelength range of this slit array can be tuned by adjusting the polarization state of the incoming light.
[1] Mayerhöfer, T.G., et al.. ACS Photonics, 2015. 2(11): p. 1567-1575.
[2] Knipper, R., et. al., in preparation.
To establish detection schemes in life science applications, specific and sensitive methods allowing for fast detection times are required. Due to the interaction of molecules with strong electromagnetic fields excited at metallic nanostructures, the molecular fingerprint specific Raman spectrum is increased by several orders of magnitude. This effect is described as surface-enhanced Raman spectroscopy (SERS) and became a very powerful analytical tool in many fields of application. Within this presentation, we will introduce innovative bottom-up strategies to prepare SERS-active nanostructures coated with a lipophilic sensor layer. To do so, the food colorant Sudan III, an indirect carcinogen substance found in chili powder, palm oil or spice mixtures, is detected quantitatively in the background of the competitor riboflavin as well as paprika powder extracts. The SERS-based detection of azorubine (E122) in commercial available beverages with different complexity (e.g. sugar content, alcohol concentration) illustrates the strong potential of SERS as a qualitative as well as semiquantitative prescan method in food analytics. Here, a good agreement between the estimated concentration employing SERS as well as the gold standard technique HPLC, a highly laborious method, is found. Finally, SERS is applied to detect vitamin B2 and B12 in cereals as well as the estimate the ratio of lycopene and β-carotene in tomatoes.
Acknowledgement: Funding the projects “QuantiSERS” and “Jenaer Biochip Initiative 2.0” within the framework “InnoProfile Transfer – Unternehmen Region“ the Federal Ministry of Education and Research, Germany (BMBF) is gratefully acknowledged.
The determination of the concentration of xenobiotics in biological matrix followed by the change of the prescribing procedure plays a major role in the transition from general to personalized medicine. For this contribution, human urine samples collected from healthy volunteers and from patients having urinary tract infection were used as biological matrix to assess the potential and limitation of LoC-SERS to detected levofloxacin and nitroxoline. The determination of both antibiotics at clinically relevant concentrations, 1.38 mM ± 0.68 mM for levofloxacin and 10-40 µM for nitroxoline, will be presented. For quantification purposes the standard addition method is combined with LoC-SERS.
Employing vibrational spectroscopy (IR-absorption and Raman spectroscopy) allows for the labelfree detection of molecular specific fingerprints of inorganic, organic and biological substances. The sensitivity of vibrational spectroscopy can be improved by several orders of magnitude via the application of plasmonic active surfaces. Within this contribution we will discuss two such approaches, namely surface enhanced Raman spectroscopy (SERS) as well as surface enhanced IR absorption (SEIRA). It will be shown that SERS using metal colloids as SERS active substrate in combination with a microfluidic lab-on-a-chip (LOC) device enables high throughput and reproducible measurements with highest sensitivity and specificity. The application of such a LOC-SERS approach for therapeutic drug monitoring (e.g. quantitative detection of antibiotics in a urine matrix) will be presented. Furthermore, we will introduce innovative bottom-up strategies to prepare SERS-active nanostructures coated with a lipophilic sensor layer as one-time use SERS substrates for specific food analysis (e.g. quantitative detection of toxic food colorants). The second part of this contribution presents a slit array metamaterial perfect absorber for IR sensing applications consisting of a dielectric layer sandwiched between two metallic layers of which the upper layer is perforated with a periodic array of slits. Light-matter interaction is greatly amplified in the slits, where also the analyte is concentrated, as the surface of the substrate is covered by a thin silica layer. Thus, already small concentrations of analytes down to a monolayer can be detected by refractive index sensing and identified by their spectral fingerprints with a standard mid-infrared lab spectrometer.
Identification of tumor and normal cells is a promising application of Raman spectroscopy. The throughput of Raman-assisted cell sorting is limited by low sensitivity. Surface-enhanced Raman spectroscopy (SERS) is a well-recognized candidate to increase the intensity of Raman signals of cells. First, different strategies are summarized to detect tumor cells using targeted SERS probes. Then, a protocol is described to prepare multicore-SERS-labels (MSLs) by aggregating gold nanoparticles, coating with a reporter molecule and a thin silver shell to further boost enhancement, encapsulating with a stable silica layer, and functionalizing by epithelial cell adhesion molecule (EpCAM) antibodies. Raman, dark field and fluorescence microscopy proved the specific and nonspecific binding of functionalized and nonfunctionalized MSLs to MCF-7 tumor cells, leukocytes from blood, and nontransformed human foreskin fibroblasts. Raman imaging and dark field microscopy indicated no uptake of MSLs, yet binding to the cellular membrane. Viability tests were performed with living tumor cells to demonstrate the low toxicity of MSL-EpCAM. The SERS signatures were detected from cells with exposure times down to 25 ms at 785-nm laser excitation. The prospects of these MSLs in multiplex assays, for enumeration and sorting of circulating tumor cells in microfluidic chips, are discussed.
We present results from the development of plasmonic chips for surface enhanced Raman spectroscopy (SERS).
Our technological approach is based on the patterning of a resist film (PMMA) on a quartz substrate by electron
beam lithography. The samples consist of periodic arrays of square shaped dots with lateral sizes around 300
nm, a period of 400 nm, and a height of 120 nm. This patterned surface is covered with a 10 nm Al2O3 film
and a 40 nm Ag film. The electromagnetic field distribution as a function of the wavelength and incidence
angle was simulated. The results show that field enhancements in the order of 10 can be achieved at 488 nm
excitation wavelength. The SERS effect of the samples was investigated experimentally using crystal violet as a
model analyte substance. The enhancement shows a good reproducibility and the values are consistent with the
simulations.
To combine fluorescence and surface enhanced Raman spectroscopy (SERS) utilizing the same plasmonic array, a silver
deposited micro fabricated quartz grating prepared by electron beam lithography is used. The most efficient Raman
signal amplification is achieved for molecules directly adsorbed on the metallic surface, while fluorescence is quenched
under these conditions. By careful adjustment, fluorescence is also enhanced which is known as metal enhanced
fluorescence. In this contribution, the combined fluorescence and SERS readout is verified by a DNA detection scheme
based on the usage of the label molecule ATTO565.
Plasmonic nanostructures exhibit a strong field enhancement due to the excitation of localized and propagating surface
plasmon polaritons. The use of these effects yields in a wide range of analytical applications. For instance, the strong
electromagnetic field enhancement may be used to dramatically increase fluorescence, Raman cross sections (surface
enhanced Raman spectroscopy - SERS) or IR absorption. Since the requirements to a powerful technique are both a
fingerprint specificity and high sensitivity, the SERS method is a powerful tool for a variety of analytical applications in
(bio)chemical and biological analysis. Because the reproducibility of established SERS arrays (e. g. roughened metal
electrodes and aggregated metal nanoparticle) across a large measuring area is rather low, we have established an e-beam
(electron beam lithography) based fabrication process yielding in regularly patterned gold nanorhomb arrays. The
anisotropic optical response of the SERS array is characterized. Furthermore, the SERS arrays are investigated with
respect to the second part of their electromagnetic enhancement, resulting in design and fabrication criteria of potential
SERS arrays.
Due to its enormous signal intensity and high fingerprint sensitivity, surface enhanced Raman spectroscopy (SERS) is a
powerful technique in chemical and biological applications. Our goal is the employment of the SERS technique for
(bio)analytical devices. A main feature in order to achieve a versatile applicability is to ensure a reproducible and
homogeneous signal across large measuring areas. Electron beam lithography is an adequate approach to assemble such
reproducible arrays. Within this contribution, the fabrication process of regular patterned nanostructures based on
electron beam lithography and argon ion beam etching is described. Using the exposure of crossed gratings of lines
within a resist layer, gold nanorhomb and nanorectangle arrays are produced on a quartz wafer. The patterns are
periodically arranged with pitches of 200 to 400 nm and exhibit sharp edges with corner radii of less than 10 nm. The
pattern dimensions in combination with the small edge radii yield a high electromagnetic field enhancement caused by
plasmonic excitation. The SERS arrays were characterized by means of SEM and were tested with respect to their SERS
response, especially with regard to their reproducibility.
Within this contribution we convincingly demonstrate that the enhancement of the intrinsically weak Raman signals
through an interaction between an analyte molecule and enhanced electromagnetic fields in the vicinity of metallic
nanostructured surfaces is an extremely potent tool in bioanalytical science because such a SERS approach comprises
high sensitivity with molecular specificity. In particular innovative approaches to realize reproducible plasmonic
nanostructures i.e. SERS substrates like e.g. lithographically produced nanostructured gold surfaces or the defined
deposition of silver nanoparticles through an enzymatic reaction are introduced.
The detection of single bacteria should be improved by lowering the acquisition time via the application of SERS
(surface enhanced Raman spectroscopy). Nano structured colloids or surfaces consisting of gold or silver can be used as
SERS active substrates. However, for biological applications mostly gold is used as SERS active substrate since silver is
toxic for bacterial cells. Furthermore, the application of gold as a SERS-active substrate allows the usage of Raman
excitation wavelengths in the red part of the electromagnetic spectrum.
For the SERS investigations on bacteria different colloids (purchased and self prepared, preaggregated and non-aggregated)
are chosen as SERS active substrates. The application of different gold colloids under gently mixing
conditions to prevent the bacterial damage allowed the recording of reproducible SERS spectra of bacteria. The SERS
spectra of B. pumilus are dominated by contributions of ingredients of the outer cell wall, e.g. the peptidoglycan layer.
SEM images of the coated bacteria demonstrate the incomplete adsorption most probably due to variations within the
binding affinities between different outer cell components and the gold colloids.
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