A Real-Time LSPR-Based Study of Metal-Organic Framework (MOF) Growth.

MOFs are known for their absorption properties and widely used for accumulation, filtering, sensorics, photothermal, catalytical and other applications. Their combination with plasmonic metal nanoparticles leads to hybrid structures that profit from the stabilizing effect and high porosity of the MOF as well as the optical and electronic properties of the nanoparticles. The growth of MOFs on plasmonic nanoparticles can be monitored in-situ using LSPR spectroscopy, simultaneously applying microfluidic reaction conditions for the fabrication of NP@MOF structures. Here, a systematic study is conducted using LSPR spectroscopy for the monitoring of the Layer-by-Layer deposition of twelve different MOFs, determining the suitability of LSPR spectroscopy for this purpose. In addition to some well-investigated materials like HKUST-1, other MOFs such as MIL-53, MIL-88 A and Cu-BDC are deposited successfully. For some MOFs such as Zn-Fum, the LSPR experiment indicates that no deposition had taken place. The results are confirmed with AFM, SEM and XPS measurements. This work shows that LSPR spectroscopy is suitable for the in-situ monitoring of LbL MOF growth and the microfluidic setup is a very promising method for the controlled manufacturing of NP@MOF hybrid structures. Further studies may include the optimization of the synthesis process or the transfer to other materials.

Isotope labeled 3D-Raman confocal imaging and atomic force microscopy study on epithelial cells interacting with the fungus Candida albicans.

The human pathogenic fungus Candida albicans damages epithelial cells during superficial infections. Here we use three-dimensional-sequential-confocal Raman spectroscopic imaging and atomic force microscopy to investigate the interaction of C. albicans wild type cells, the secreted C. albicans peptide toxin candidalysin and mutant cells lacking candidalysin with epithelial cells. The candidalysin is responsible for epithelial cell damage and exhibits in its deuterated form an identifiable Raman signal in a frequency region distinct from the cellular frequency region. Vibration modes at 2100-2200 cm-1 attributed to carbon­deuterium bending and at 477 cm-1, attributed to the nitrogen­deuterium out-of-plane bending, found around the nucleus, can be assigned to deuterated candidalysin. Atomic force microscopy visualized 100 nm deep lesions on the cell and force-distance curves indicate the higher adhesion on pore surrounding after incubation with candidalysin. Candidalysin targets the plasma membrane, but is also found inside of the cytosol of epithelial cells during C. albicans infection.

LSPR-Based Biosensing Enables the Detection of Antimicrobial Resistance Genes.

The development of rapid, simple, and accurate bioassays for the detection of nucleic acids has received increasing demand in recent years. Here, localized surface plasmon resonance (LSPR) spectroscopy for the detection of an antimicrobial resistance gene, sulfhydryl variable ß-lactamase (blaSHV), which confers resistance against a broad spectrum of ß-lactam antibiotics is used. By performing limit of detection experiments, a 23 nucleotide (nt) long deoxyribonucleic acid (DNA) sequence down to 25 nm was detected, whereby the signal intensity is inversely correlated with sequence length (23, 43, 63, and 100 nt). In addition to endpoint measurements of hybridization events, the setup also allowed to monitor the hybridization events in real-time, and consequently enabled to extract kinetic parameters of the studied binding reaction. Performing LSPR measurements using single nucleotide polymorphism (SNP) variants of blaSHV revealed that these sequences can be distinguished from the fully complementary sequence. The possibility to distinguish such sequences is of utmost importance in clinical environments, as it allows to identify mutations essential for enzyme function and thus, is crucial for the correct treatment with antibiotics. Taken together, this system provides a robust, label-free, and cost-efficient analytical tool for the detection of nucleic acids and will enable the surveillance of antimicrobial resistance determinants.

Microfluidic-Generated Seeds for Gold Nanotriangle Synthesis in Three or Two Steps.

Nanoparticle synthesis has drawn great attention in the last decades. The study of crystal growth mechanisms and optimization of the existing methods lead to the increasing accessibility of nanomaterials, such as gold nanotriangles which have great potential in the fields of plasmonics and catalysis. To form such structures, a careful balance of reaction parameters has to be maintained. Herein, a novel synthesis of gold nanotriangles from seeds derived with a micromixer, which provides a highly efficient mixing and simple parameter control is reported. The impact of the implemented reactor on the primary seed characteristics is investigated. The following growth steps are studied to reveal the phenomena affecting the shape yield. The use of microfluidic seeds led to the formation of well-defined triangles with a narrower size distribution compared to the entirely conventional batch synthesis. A shortened two-step procedure for the formation of triangles directly from primary seeds, granting an express but robust synthesis is further described. Moreover, the need for a thorough study of seed crystallinity depending on the synthesis conditions, which - together with additional parameter optimization - will bring a new perspective to the use of micromixers which are promising for scaling up nanomaterial production is highlighted.

Optical chemosensors for environmental monitoring of toxic metals related to Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia and progresses from mild memory loss to severe decline in thinking, behavioral and social skills, which dramatically impairs a person's ability to function independently. Genetics, some health disorders and lifestyle have all been connected to AD. Also, environmental factors are reported as contributors to this illness. The presence of heavy metals in air, water, food, soil and commercial products has increased tremendously. Accumulation of heavy metals in the body leads to serious malfunctioning of bodily organs, specifically the brain. For AD, a wide range of heavy metals have been reported to contribute to its onset and progression and the manifestation of its hallmarks. In this review, we focus on detection of highly toxic heavy metals such as mercury, cadmium, lead and arsenic in water. The presence of heavy metals in water is very troubling and regular monitoring is warranted. Optical chemosensors were designed and fabricated for determination of ultra-trace quantities of heavy metals in water. They have shown advantages when compared to other sensors, such as selectivity, low-detection limit, fast response time, and wide-range determination under optimal sensing conditions. Therefore, implementing optical chemosensors for monitoring levels of toxic metals in water represents an important contribution in fighting AD.

Fast detection of bacterial contamination in fresh produce using FTIR and spectral classification.

Screening for microbial contaminants in fresh produce is a lengthy process relative to their short shelf-life. The aim of this study is to develop a comprehensive assay which employs FTIR and spectral classification algorithm for detection of bacterial contamination of fresh produce. The procedure starts by soaking a sample of the fresh produce in broth for 5 h. Then, magnetic nanoparticles are added to capture bacteria which are then collected and prepared for FTIR scanning. The generated FTIR spectra are compared against an in-house database of different bacterial species (n = 6). The ability of the database to discriminate contaminated and uncontaminated samples and to identify the bacterial species was assessed. The compatibility of the FTIR procedures with subsequent DNA extraction and PCR was tested. The developed procedure was applied for assessment of bacterial contamination in fresh produce samples from the market (n = 78). Results were compared to the conventional culture methods. The generated FTIR database coupled to spectral classification was able to detect bacterial contamination with overall accuracy exceeding 90%. The sample processing did not alter the integrity of the bacterial DNA which was suitable for PCR. On application to fresh produce samples collected from the market, the developed method was able to detect bacterial contamination with 94% concordance with the culture method. In conclusion, the developed procedure can be applied for fast detection of microbial contamination in fresh produce with comparable accuracy to conventional microbiological assays and is compatible with subsequent molecular assays.

Sequence Effect on the Activity of DNAzyme with Covalently Attached Hemin and Their Potential Bioanalytical Application.

In this work we investigated the effect of a DNA oligonucleotide sequence on the activity of a DNAzyme with covalently attached hemin. For this purpose, we synthesized seven DNA-hemin conjugates. All DNA-hemin conjugates as well as DNA/hemin complexes were characterized using circular dichroism, determination of melting temperatures and pKa of hemin. We observed that hemin conjugation in most cases led to the formation of parallel G-quadruplexes in the presence of potassium and increased thermal stability of all studied systems. Although the activity of DNA-hemin conjugates depended on the sequence used, the highest activity was observed for the DNA-hemin conjugate based on a human telomeric sequence. We used this DNAzyme for development of "sandwich" assay for detection of DNA sequence. For this assay, we used electric chip which could conduct electricity after silver deposition catalyzed by DNAzyme. This method was proved to be selective towards DNA oligonucleotides with mismatches and could be used for the detection of the target. To prove the versatility of our DNAzyme probe we also performed experiments with streptavidin-coated microplates. Our research proved that DNAzyme with covalently attached hemin can be used successfully in the development of heterogeneous assays.

The effect of layer thickness and immobilization chemistry on the detection of CRP in LSPR assays.

The immobilization of a capture molecule represents a crucial step for effective usage of gold nanoparticles in localized surface plasmon resonance (LSPR)-based bioanalytics. Depending on the immobilization method used, the resulting capture layer is of varying thickness. Thus, the target binding event takes place at different distances to the gold surface. Using the example of a C-reactive protein immunoassay, different immobilization methods were tested and investigated with regard to their resulting target signal strength. The dependency of the target signal on the distance to the gold surface was investigated utilizing polyelectrolyte bilayers of different thickness. It could be experimentally demonstrated how much the LSPR-shift triggered by a binding event on the gold nanoparticles decreases with increasing distance to the gold surface. Thus, the sensitivity of an LSPR assay is influenced by the choice of immobilization chemistry.

Review: tomographic imaging flow cytometry.

Within the last decades, conventional flow cytometry (FC) has evolved as a powerful measurement method in clinical diagnostics, biology, life sciences and healthcare. Imaging flow cytometry (IFC) extends the power of traditional FC by adding high resolution optical and spectroscopic information. However, the conventional IFC only provides a 2D projection of a 3D object. To overcome this limitation, tomographic imaging flow cytometry (tIFC) was developed to access 3D information about the target particles. The goal of tIFC is to visualize surfaces and internal structures in a holistic way. This review article gives an overview of the past and current developments in tIFC.

Time Optimization of Seed-Mediated Gold Nanotriangle Synthesis Based on Kinetic Studies.

The synthesis of shape-anisotropic plasmonic nanoparticles such as gold nanotriangles is of increasing interest. These particles have a high potential for applications due to their notable optical properties. A key challenge of the synthesis is usually the low reproducibility. Even the optimized seed-based methods often lack in the synthesis yield or are labor- and time-consuming. In this work, a seed-mediated synthesis with high reproducibility is replicated in order to determine the necessary reaction time for each step. Online monitoring of the reaction mixtures by UV-VIS spectroscopy is used as a powerful tool to track the evolution of the synthesis. The kinetics of the individual stages is elucidated by real-time investigations. As a consequence, the complete synthesis could be optimized and can now be realized in a single day instead of three without any loss in the resulting sample quality.

Modification of Surface Bond Au Nanospheres by Chemically and Plasmonically Induced Pd Deposition.

In this work we investigated methods of modifying gold nanospheres bound to a silicon surface by depositing palladium onto the surfaces of single nanoparticles. Bimetallic Au-Pd nanoparticles can thus be gained for use in catalysis or sensor technology. For Pd deposition, two methods were chosen. The first method was the reduction of palladium acetate by ascorbic acid, in which the amounts of palladium acetate and ascorbic acid were varied. In the second method we utilized light-induced metal deposition by making use of the plasmonic effect. Through this method, the surface bond nanoparticles were irradiated with light of wavelengths capable of inducing plasmon resonance. The generation of hot electrons on the particle surface then reduced the palladium acetate in the vicinity of the gold nanoparticle, resulting in palladium-covered gold nanospheres. In our studies we demonstrated the effect of both enhancement methods by monitoring the particle heights over enhancement time by atomic force microscopy (AFM), and investigated the influence of ascorbic acid/Pd acetate concentration as well as the impact of the irradiated wavelengths on the enhancement effect. It could thus be proven that both methods were valid for obtaining a deposition of Pd on the surface of the gold nanoparticles. Deposition of Pd on the gold particles using the light-assisted method could be observed, indicating the impact of the plasmonic effect and hot electron for Pd acetate reduction on the gold particle surface. In the case of the reduction method with ascorbic acid, in addition to Pd deposition on the gold nanoparticle surface, larger pure Pd particles and extended clusters were also generated. The reduction with ascorbic acid however led to a considerably thicker Pd layer of up to 54 nm in comparison to up to 11 nm for the light-induced metal deposition with light resonant to the particle absorption wavelength. Likewise, it could be demonstrated that light of non-resonant wavelengths was not capable of initiating Pd deposition, since a growth of only 1.6 nm (maximum) was observed for the Pd layer.

Application of Localized Surface Plasmon Resonance Spectroscopy to Investigate a Nano-Bio Interface.

The accurate determination of events at the interface between a biological system and nanomaterials is necessary for efficacy and safety evaluation of novel nano-enabled medical products. Investigating the interaction of proteins with nanoparticles (NPs) and the formation of protein corona on nanosurfaces is particularly challenging from the methodological point of view due to the multiparametric complexity of such interactions. This study demonstrated the application of localized surface plasmon resonance (LSPR) spectroscopy as a low-cost and rapid biosensing technique that can be used in parallel with other sophisticated methods to monitor nano-bio interplay. Interaction of citrate-coated gold NPs (AuNPs) with human plasma proteins was selected as a case study to evaluate the applicability and value of scientific data acquired by LSPR as compared to fluorescence spectroscopy, which is one of the most used techniques to study NP interaction with biomolecules. LSPR results obtained for interaction of AuNPs with bovine serum albumin, glycosylated human transferrin, and non-glycosylated recombinant human transferrin correlated nicely with the adsorption constants obtained by fluorescence spectroscopy. This ability, complemented by its fast operation and reliability, makes the LSPR methodology an attractive option for the investigation of a nano-bio interface.

A miRNA biosensor based on localized surface plasmon resonance enhanced by surface-bound hybridization chain reaction.

The dysregulation of the concentration of individual circulating microRNAs or small sets of them has been recognized as a marker of disease. For example, an increase of the concentration of circulating miR-17 has been linked to lung cancer and metastatic breast cancer, while its decrease has been found in multiple sclerosis and gastric cancer. Consequently, techniques for the fast, specific and simple quantitation of microRNAs are becoming crucial enablers of early diagnosis and therapeutic follow-up. DNA based biosensors can serve this purpose, overcoming some of the drawbacks of conventional lab-based techniques. Herein, we report a cost-effective, simple and robust biosensor based on localized surface plasmon resonance and hybridization chain reaction. Immobilized gold nanoparticles are used for the detection of miR-17. Specificity of the detection was achieved by the use of hairpin surface-tethered probes and the hybridization chain reaction was used to amplify the detection signal and thus extend the dynamic range of the quantitation. Less than 1 h is needed for the entire procedure that achieved a limit of detection of about 1 pM or 50 amol/measurement, well within the reported useful range for diagnostic applications. We suggest that this technology could be a promising substitute of traditional lab-based techniques for the detection and quantification of miRNAs after these are extracted from diagnostic specimens and their analysis is thus made possible.

DNA-Biofunctionalization of CTAC-Capped Gold Nanocubes.

Clinical diagnostics and disease control are fields that strongly depend on technologies for rapid, sensitive, and selective detection of biological or chemical analytes. Nanoparticles have become an integral part in various biomedical detection devices and nanotherapeutics. An increasing focus is laid on gold nanoparticles as they express less cytotoxicity, high stability, and hold unique optical properties with the ability of signal transduction of biological recognition events with enhanced analytical performance. Strong electromagnetic field enhancements can be found in close proximity to the nanoparticle that can be exploited to enhance signals for e.g., metal-enhanced fluorescence or Raman spectroscopy. Even stronger field enhancements can be achieved with sharp-edged nanoparticles, which are synthesized with the help of facet blocking agents, such as cetyltrimethylammonium bromide/chloride (CTAB/CTAC). However, chemical modification of the nanoparticle surface is necessary to reduce the particle's cytotoxicity, stabilize it against aggregation, and to bioconjugate it with biomolecules to increase its biocompatibility and/or specificity for analytical applications. Here, a reliable two-step protocol following a ligand exchange with bis (p-sulfonatophenyl) phenyl phosphine (BSPP) as the intermediate capping-agent is demonstrated, which results in the reliable biofunctionalization of CTAC-capped gold nanocubes with thiol-modified DNA. The functionalized nanocubes have been characterized regarding their electric potential, plasmonic properties, and stability against high concentrations of NaCl and MgCl2.

Loop-mediated amplification as promising on-site detection approach for Legionella pneumophila and Legionella spp.

Recently Legionella pneumophila is the main causative waterborne organism of severe respiratory infections. Additionally, other Legionella species are documented as human pathogens. In our work, we describe a rapid detection method which combines two advantages for sensitive and specific detection of the genus Legionella: the fast isothermal amplification method "Loop-mediated isothermal AMPlification" (LAMP), and a colorimetric detection method using the metal indicator hydroxynaphtol blue (HBN) which allows to determine an optical signal with a simple readout (with the naked eye). Moreover, we present two approaches for minimizing the assay volume using a stationary microchip LAMP and droplet digital-based LAMP (ddLAMP) as promising highly sensitive setups.

Time-Resolved Study of Site-Specific Corrosion in a Single Crystalline Silver Nanoparticle.

We followed over 24 h a corrosion process in monocrystalline triangular-shaped nanoparticles at a single-particle level by atomic force microscopy and optical spectroscopy techniques under ambient laboratory conditions. The triangular-shaped form of the particles was selected, because the crystallographic orientation of the particles is well defined upon their deposition on a substrate. We observed that the particles already start to alter within this time frame. Surprisingly, the corrosion starts predominantly from the tips of the particles and it creates within few hours large protrusions, which strongly suppress the plasmon character of the particles. These observations support the crystallographic model of these particles consisting of a high-defect hexagonal closed packed layer, and they could help material scientists to design more stable silver nanoparticles. Moreover, this described technique can be used to reveal kinetics of the corrosion in the nanoscale of other materials.

Fabrication of micro-patterned substrates for plasmonic sensing by piezo-dispensing of colloidal nanoparticles.

In this work we describe a very fast and flexible method for fabrication of plasmon-supporting substrates with micro-patterning capability, which is optimized for plasmonic sensing. We combined a wet chemistry approach to synthesize metallic nanoparticles with a piezo-dispensing system enabling deposition of nanoparticles on the substrates with micrometer precision. In this way, an arbitrary pattern consisting of 200 µm small spots containing plasmonic nanostructures can be produced. Patterns with various nanoparticles exhibiting different plasmonic properties were combined, and the surface density of the particles could be easily varied via their solution concentrations. We showed that under controlled conditions the dispensing process caused no aggregation of the particles and it enabled full transfer of the colloidal solutions onto the substrate. This is an important condition, which enables these substrates to be used for reliable plasmonic sensing based on monitoring the spectral shift of the nanoparticles. We demonstrated the functionality of such substrates by detection of small protein adsorption on the spots based on plasmon label-free sensing method.

Plasmonic Nanosensor Array for Multiplexed DNA-based Pathogen Detection.

In this research we introduce a plasmonic nanoparticle based optical biosensor for monitoring of molecular binding events. The sensor utilizes spotted gold nanoparticle arrays as sensing platform. The nanoparticle spots are functionalized with capture DNA sequences complementary to the analyte (target) DNA. Upon incubation with the target sequence, it will bind on the respectively complementary functionalized particle spot. This binding changes the local refractive index, which is detected spectroscopically as the resulting changes of the localized surface plasmon resonance (LSPR) peak wavelength. In order to increase the signal, a small gold nanoparticle label is introduced. The binding can be reversed using chemical means (10 mM HCl). It is demonstrated that multiplexed detection and identification of several fungal pathogen DNA sequences subsequently on one sensor array are possible by this approach.

AFM-Based Probing of the Flexibility and Surface Attachment of Immobilized DNA Origami.

The flexible and precise immobilization of self-organizing DNA nanostructures represents a key step in the integration of DNA-based material for potential electronic or sensor applications. However, the involved processes have still not been well studied and are not yet fully understood. Thus, we investigated the potential for the mechanical manipulation of DNA origami by atomic force microscopy (AFM) in order to study the interaction between intramolecular flexibility and surface-attachment forces. AFM is particularly suitable for nanoscale manipulation. Previous studies showed the potential for pushing, bending, and cutting double-stranded DNA (dsDNA) with an AFM tip. Understanding the involved parameters may enable control over different processes such as nanointegration, precise cutting, and stretching of preassembled DNA origami. We demonstrate the defined manipulation and flexibility of DNA origami immobilized on mica in the nanometer range: controlled cutting, folding, and stretching as a function of the magnesium concentration.

LSPR Detection of Nucleic Acids on Nanoparticle Monolayers.

Noble metal nanoparticles are well known for their unique optical properties. Density oscillations of the nanoparticle conduction electrons are induced at a specific frequency by an external incident light beam. This phenomenon is known under the term localized surface plasmon resonance (LSPR). The spectral position of the resonance band is determined by shape, size, and material of the nanoparticle and influenced by changes of the local refractive index of the surrounding medium. The latter gives the opportunity to use noble metal nanoparticles as label-free bioanalytical sensors. Biomolecules can be bound directly on the nanoparticle surface, which leads to a change of the local refractive index, and a shift of the peak maximum is detected by absorbance spectroscopy. This method is used for bioanalytical diagnostics. Here, a DNA sensing protocol for real-time measurements in situ using this system will be presented. A dense layer of noble metal nanoparticles is immobilized on a glass substrate and implemented in a microfluidic chamber, where the spectroscopic measurements are conducted.