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.

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.

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.

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.

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.

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.

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.

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.

A New Strategy for Silver Deposition on Au Nanoparticles with the Use of Peroxidase-Mimicking DNAzyme Monitored via a Localized Surface Plasmon Resonance Technique.

Peroxidase-mimicking DNAzyme was applied as a catalyst of silver deposition on gold nanoparticles. This DNAzyme is formed when hemin binds to the G-quadruplex-forming DNA sequence. Such a system is able to catalyze a redox reaction with a one- or two-electron transfer. The process of silver deposition was monitored via a localized surface plasmon resonance technique (LSPR), which allows one to record scattering spectrum of a single nanoparticle. Our study showed that DNAzyme is able to catalyze silver deposition. The AFM experiments proved that DNAzyme induced the deposition of silver shells of approximately 20 nm thickness on Au nanoparticles (AuNPs). Such an effect is not observed when hemin is absent in the system. However, we noticed non-specific binding of hemin to the capture oligonucleotides on a gold NP probe that also induced some silver deposition, even though the capture probe was unable to form G-quadruplex. Analysis of SEM images indicated that the surface morphology of the silver layer deposited by DNAzyme is different from that obtained for hemin alone. The proposed strategy of silver layer synthesis on gold nanoparticles catalyzed by DNAzyme is an innovative approach and can be applied in bioanalysis (LSPR, electrochemistry) as well as in material sciences.

Surface Mobility and Ordered Rearrangement of Immobilized DNA Origami.

The immobilization of DNA nanostructures on a surface is a key step for the integration of DNA-based material in nanotechnology for electronic or sensorial applications. Thereby the arrangement and distribution at the substrate surface is of growing interest. Top-down approaches such as lithography have been reported, but show certain limitations regarding costs and/or throughput. Here we present a self-assembly effect observed when already immobilized and dried origami preparations were again rehydrated under certain conditions, resulting in a certain ordering of densely packed origami structures. We investigate the influence of different parameters in order to reveal the underlying mechanisms, and find that subsequent droplet formation and interfacial tension during the droplet drying play a role. Further adjustment of these parameters could develop the introduced effect to an additional tool for controlled integration of DNA nanostructures.

Localized surface plasmon resonance (LSPR) biosensing using gold nanotriangles: detection of DNA hybridization events at room temperature.

We present a proof-of-concept of the application of gold nanotriangles in sequence specific DNA detection, using localized surface plasmon resonance (LSPR) spectroscopy and dark-field optical microscopy. The sensing platform comprises gold nanotriangles immobilized on a glass chip and oligonucleotides as probes. Probe formation and testing complementary and non-complementary targets followed common chip technology protocols. Gold nanotriangles showed a remarkable sensitivity of 468 nm per RIU and allowed detection of 20-mer targets. When the target sequence was part of a 50-mer synthetic DNA oligonucleotide, LSPR shifts as high as 35 nm were observed. Conversely, when the target was present in PCR products of ca. 350 bp, obtained from clinical samples, LSPR shifts larger than 20 nm were observed. Moreover, LSPR shifts were less than ±1 nm for the respective non-complementary targets. These results with gold nanotriangles as sensors are a notable improvement to the LSPR shifts of less than 5 nm usually obtained for spherical gold nanoparticles of comparable sizes. Optimal conditions for the detection of synthetic and PCR product targets using gold nanotriangles and oligonucleotide probes were achieved with low percentages of intercalating thioalkanes; target hybridization at room temperature, 3 hours of incubation, and 2× SSC buffer stringency conditions.

Combined dielectrophoresis-Raman setup for the classification of pathogens recovered from the urinary tract.

Rapid and effective methods of pathogen identifications are of major interest in clinical microbiological analysis to administer timely tailored antibiotic therapy. Raman spectroscopy as a label-free, culture-independent optical method is suitable to identify even single bacteria. However, the low bacteria concentration in body fluids makes it difficult to detect their characteristic molecular fingerprint directly in suspension. Therefore, in this study, Raman spectroscopy is combined with dielectrophoresis, which enables the direct translational manipulation of bacteria in suspensions with spatial nonuniform electrical fields so as to perform specific Raman spectroscopic characterization. A quadrupole electrode design is used to capture bacteria directly from fluids in well-defined microsized regions. With live/dead fluorescence viability staining, it is verified, that the bacteria survive this procedure for the relevant range of field strengths. The dielectrophoretic enrichment of bacteria allows for obtaining high quality Raman spectra in dilute suspensions with an integration time of only one second. As proof-of-principle study, the setup was tested with Escherichia coli and Enterococcus faecalis, two bacterial strains that are commonly encountered in urinary tract infections. Furthermore, to verify the potential for dealing with real world samples, pathogens from patients' urine have been analyzed. With the additional help of multivariate statistical analysis, a robust classification model could be built and allowed the classification of those two strains within a few minutes. In contrast, the standard microbiological diagnostics are based on very time-consuming cultivation tests. This setup holds the potential to reduce the crucial parameter diagnosis time by orders of magnitude.

Functionalization of Phosphate and Tellurite Glasses and Spherical Whispering Gallery Mode Microresonators.

Active whispering gallery mode resonators made as spherical microspheres doped with quantum dots or rare earth ions achieve high quality factors and are excellent candidates for biosensors capable of detecting biomolecules at low concentrations. However, to produce quantum dot-doped microspheres, new low melting temperature glasses are sought, which require surface functionalization and antibody immobilization for biosensor development. Here, we demonstrate the successful functionalization of three low melting point glasses and microspheres made of them. The glasses were made from sodium borophosphate, sodium aluminophosphate, and tellurite, and then, they were functionalized using (3-glycidyloxypropyl)trimethoxysilane in ethanol- and toluene-based protocols. Proper silanization was confirmed by energy-dispersive X-ray spectroscopy and fluorescence microscopy of an amino-modified luminescent oligonucleotide probe. Fluorescence imaging showed successful silanization for all tested samples and no degradation for aluminophosphate and tellurite glasses. The strongest signal was registered for tellurite glass samples functionalized using the toluene-based silanization protocol. This conclusion implies that this functionalization method is the most efficient and is highly recommended for future antibody immobilization and biosensing application.

The invention of immersion ultramicroscopy in 1912-the birth of nanotechnology?

Dawn of nanotechnology: the immersion ultramicroscope was patented a century ago. When an analyte was examined with an antique instrument and with state-of-the-art technology, the historic assumptions were confirmed: the size and shape of the nanoparticles are in the same range as that described 100 years ago. The spectra of the Tyndall cones caused by the shape of the nanoparticles were also described correctly-long before electron microscopy was able to image single nanoparticles.

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.

Sensoric potential of gold-silver core-shell nanoparticles.

The sensitivities of five different core-shell nanostructures were investigated towards changes in the refractive index of the surrounding medium. The shift of the localized surface plasmon resonance (LSPR) maximum served as a measure of the (respective) sensitivity. Thus, gold-silver core-shell nanoparticles (NPs) were prepared with different shell thicknesses in a two-step chemical process without the use of any (possibly disturbing) surfactants. The measurements were supported by ultramicroscopic images in order to size the resulting core-shell structures. When compared to sensitivities of nanostructures reported in the literature with those of the (roughly spherical) gold-silver core-shell NPs, the latter showed comparable (or even higher) sensitivities than gold nanorods. The experimental finding is supported by theoretical calculation of optical properties of such core-shell NP. Extinction spectra of ideal spherical and deformed core-shell NPs with various core/shell sizes were calculated, and the presence of an optimal silver shell thickness with increased sensitivity was confirmed. This effect is explained by the existence of two overlapping plasmon bands in the NP, which change their relative intensity upon change of refractive index. Results of this research show a possibility of improving LSPR sensor by adding an extra metallic layer of certain thickness.

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.

Nanoparticle layer deposition for plasmonic tuning of microstructured optical fibers.

Plasmonic nanoparticles with spectral properties in the UV-to-near-IR range have a large potential for the development of innovative optical devices. Similarly, microstructured optical fibers (MOFs) represent a promising platform technology for fully integrated, next-generation plasmonic devices; therefore, the combination of MOFs and plasmonic nanoparticles would open the way for novel applications, especially in sensing applications. In this Full Paper, a cost-effective, innovative nanoparticle layer deposition (NLD) technique is demonstrated for the preparation of well-defined plasmonic layers of selected particles inside the channels of MOFs. This dynamic chemical deposition method utilizes a combination of microfluidics and self-assembled monolayer (SAM) techniques, leading to a longitudinal homogeneous particle density as long as several meters. By using particles with predefined plasmonic properties, such as the resonance wavelength, fibers with particle-adequate spectral characteristics can be prepared. The application of such fibers for refractive-index sensing yields a sensitivity of about 78 nm per refractive index unit (RIU). These novel, plasmonically tuned optical fibers with freely selected, application-tailored optical properties present extensive possibilities for applications in localized surface plasmon resonance (LSPR) sensing.

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.