Nanospray-assisted deposition of silver nanoparticles for mapping of a peptide in nanofibrous layers via surface-enhanced Raman spectrometry.

Surface-enhanced Raman spectrometry (SERS) is a universal detection tool identifying molecules via vibrations of their chemical bonds. Its function requires the close localization of metal nanostructures and the analyte. In this work, we present a lab-made instrumentation for the deposition of silver nanoparticles on a strongly hydrophilic nanofibrous composite via a nanospray for SERS mapping of an incorporated peptide. The nanospray-sample distance was revealed as the most crucial parameter since it directly influences the moisture of the deposited colloid. Residual water was recognized as a sensitivity enhancer. Additionally, we continuously introduced a solution of sodium chloride to the colloid increasing its ionic strength, which formed a more homogeneous profile of the deposit. After the deposition process, the treated sample was scanned via a SERS laser and the collected Raman spectra were transformed into a distribution map of the peptide at a concentration of 5 µg/g.

Microfluidic device for concentration and SERS-based detection of bacteria in drinking water.

There is a constant need for the development of easy-to-operate systems for the rapid and unambiguous identification of bacterial pathogens in drinking water without the requirement for time-consuming culture processes. In this study, we present a disposable and low-cost lab-on-a-chip device utilizing a nanoporous membrane, which connects two stacked perpendicular microfluidic channels. Whereas one of the channels supplies the sample, the second one attracts it by potential-driven forces. Surface-enhanced Raman spectrometry (SERS) is employed as a reliable detection method for bacteria identification. To gain the effect of surface enhancement, silver nanoparticles were added to the sample. The pores of the membrane act as a filter trapping the bodies of microorganisms as well as clusters of nanoparticles creating suitable conditions for sensitive SERS detection. Therein, we focused on the construction and characterization of the device performance. To demonstrate the functionality of the microfluidic chip, we analyzed common pathogens (Escherichia coli DH5α and Pseudomonas taiwanensis VLB120) from spiked tap water using the optimized experimental parameters. The obtained results confirmed our system to be promising for the construction of a disposable optical platform for reliable and rapid pathogen detection which couples their electrokinetic concentration on the integrated nanoporous membrane with SERS detection.

Electrospray: More than just an ionization source.

Electrospraying (ES) is a potential-driven process of liquid atomization, which is employed in the field of analytical chemistry, particularly as an ionization technique for mass spectrometric analyses of biomolecules. In this review, we demonstrate the extraordinary versatility of the electrospray by overviewing the specifics and advanced applications of ES-based processing of low molecular mass compounds, biomolecules, polymers, nanoparticles, and cells. Thus, under suitable experimental conditions, ES can be used as a powerful tool for highly controlled deposition of homogeneous films or various patterns, which may sometimes even be organized into 3D structures. We also emphasize its capacity to produce composite materials including encapsulation systems and polymeric fibers. Further, we present several other, less common ES-based applications. This review provides an insight into the remarkable potential of ES, which can be very useful in the designing of innovative and unique strategies.

Bi-Ligand Modification of Nanoparticles: An Effective Tool for Surface-Enhanced Raman Spectrometry in Salinated Environments.

Elimination of massive aggregation of nanoparticles in the sample of high ionic strength is a prerequisite for the sensitive analysis through a surface-enhanced Raman spectrometry (SERS). We present a system of silver colloid modification composed of two thiolated modifiers (3-mercaptopropionic acid and thiolated polyethylene glycol) both creating a strong Ag-S bond. At their optimal molar ratio, the polymer acts as a steric barrier preventing direct nanoparticle-nanoparticle interaction, while the low-molecular organic acid creates areas accessible for the analyte molecules. Thus, this approach is an excellent tool for sustaining both the colloidal stability and SERS sensitivity. The functionality of the system was demonstrated on the SERS analysis of myoglobin from a saline solution. The favorable creation of hot spots was achieved by laser-induced sintering.

Combination of liquid-based column separations with surface-enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy is a constantly developing analytical method providing not only high-sensitive quantitative but also qualitative information on an analyte. Thus, it is reasonable that it has been tested as a promising detection method in column separations. Although its implementation in analytical separations is not widespread, some surprising results, like enormous signal enhancement and demonstrations of single-molecule identifications, proved in only a few special examples, indicate the potential of the method. The high detection sensitivity and selectivity would be of paramount importance in trace analyses of biologically relevant molecules in complex matrices. However, the combination of surface-enhanced Raman spectroscopy with column separation methods brings two principal issues. Interactions of analytes with metal substrates can cause deteriorations of separations and the detection can be affected by background electrolytes or elution agents. Thus, in principle, this review is on the experimental and methodological solutions to these problems. First, theoretical and practical aspects of Raman scattering, and excitation of surface plasmon in colloid suspensions of nanoparticles and on planar nanostructured substrates are briefly explained. Advances in experimental arrangements of on-line and at-line couplings with column liquid phase separation methods, including microfluidic devices, are described together with chosen analytical applications.

Surface enhanced Raman spectroscopy in microchip electrophoresis.

Coupling microchip capillary electrophoresis to surface enhanced Raman spectroscopy (MCE-SERS) combines the high separation power of capillary electrophoresis with the capability to obtain vibrational fingerprint spectra for compound identification. Raman spectroscopy is a structurally descriptive and label-free detection method which is particularly suited for chemical analysis because it is non-destructive and allows the identification of analytes. However, it suffers from poor sensitivity and sometimes even requires acquisition times far longer than the typical peak width of electrophoretic separations. The Raman intensity can be drastically improved if the analyte is brought into close proximity to nanostructured metal surfaces or colloids due to the surface enhancement effect. This paper presents a novel approach in the field of MCE-SERS on-line coupling. The key element of the developed glass microfluidic device is a dosing structure which consists of two side channels joining the MCE channel symmetrically after the electrophoretic separation of the analytes. The dosing channel supplies silver nanoparticles (Ag-NPs), to the separated electrophoretic zones which facilitates an on-the-fly recording of SERS-spectra of the separated compounds. The functionality of the MCE-SERS chip was evaluated by the analysis of a rhodamine model mixture within 90 s achieving RSD of migration times below 1.5%. The approach was successfully applied for the analysis of the food additive riboflavin in a barbecue sauce.

Recent strategies toward microfluidic-based surface-enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) is an extremely powerful analytical tool, which not only yields information about the molecular structure of the analyte in the form of characteristic vibrational spectrum but also gives sensitivities approaching those in fluorescence spectroscopy. The SERS measurement on the microfluidic platform provides possibility to manufacture the device with design perfectly fulfilling the needs of the application with minimal sample consumption. This review aims at describing basic strategies for SERS measurement in microfluidic devices published in the last decade and covers current trends in microfluidics with SERS detection in the field of bioanalysis and approaches toward on-line coupling of liquid-based separation techniques with SERS detection.

Recent advances in CE-MS coupling: Instrumentation, methodology, and applications.

This review focuses on the latest development of microseparation electromigration methods in capillaries and microfluidic devices coupled with MS for detection and identification of important analytes. It is a continuation of the review article on the same topic by Kleparnik (Electrophoresis 2015, 36, 159-178). A wide selection of 161 relevant articles covers the literature published from June 2014 till May 2016. New improvements in the instrumentation and methodology of MS interfaced with capillary or microfluidic versions of zone electrophoresis, isotachophoresis, and isoelectric focusing are described in detail. The most frequently implemented MS ionization methods include electrospray ionization, matrix-assisted desorption/ionization and inductively coupled plasma ionization. Although the main attention is paid to the development of instrumentation and methodology, representative examples illustrate also applications in the proteomics, glycomics, metabolomics, biomarker research, forensics, pharmacology, food analysis, and single-cell analysis. The combinations of MS with capillary versions of electrochromatography, and micellar electrokinetic chromatography are not included.

Interface-free capillary electrophoresis-mass spectrometry system with nanospray ionization-Analysis of dexrazoxane in blood plasma.

The newly developed interface-free capillary electrophoresis-nanospray/mass spectrometry system (CE-nESI/MS) was applied for rapid analysis of the cardioprotective drug dexrazoxane and its hydrolysed form ADR-925 in deproteinized blood plasma samples. The aim of this study was to test the simplest possible CE-nESI/MS instrumentation for analyses of real samples. This interface-free system, utilizing single piece of a narrow bore capillary as both the electrophoretic separation column and the nanospray emitter, was operated at a flow rate of 30nL/min. Excellent electrophoretic separation and sensitive nanospray ionization was achieved with the use of only one high voltage power supply. In addition, hydrophobic external coating was developed and tested for additional stability of the nanospray ionization. To our knowledge this is the first study devoted to the analysis of dexrazoxane and ADR-925 by capillary electrophoresis-mass spectrometry.

The "putative" role of transcription factors from HlWRKY family in the regulation of the final steps of prenylflavonid and bitter acids biosynthesis in hop (Humulus lupulus L.).

Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.

Reproducible preparation of nanospray tips for capillary electrophoresis coupled to mass spectrometry using 3D printed grinding device.

The use of high quality fused silica capillary nanospray tips is critical for obtaining reliable and reproducible electrospray/MS data; however, reproducible laboratory preparation of such tips is a challenging task. In this work, we report on the design and construction of low-cost grinding device assembled from 3D printed and commercially easily available components. Detailed description and characterization of the grinding device is complemented by freely accessible files in stl and skp format allowing easy laboratory replication of the device. The process of sharpening is aimed at achieving maximal symmetricity, surface smoothness and repeatability of the conus shape. Moreover, the presented grinding device brings possibility to fabricate the nanospray tips of desired dimensions regardless of the commercial availability. On several samples of biological nature (reserpine, rabbit plasma, and the mixture of three aminoacids), performance of fabricated tips is shown on CE coupled to MS analysis. The special interest is paid to the effect of tip sharpness.

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme.

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

Computational exploration of microRNAs from expressed sequence tags of Humulus lupulus, target predictions and expression analysis.

Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental-in silico computational based approach was adopted for the identification and characterization of miRNAs in Humulus lupulus (hop), which is widely cultivated for use by the brewing industry and apart from, used as a medicinal herb. A total of 22 miRNAs belonging to 17 miRNA families were identified in hop following comparative computational approach and EST-based homology search according to a series of filtering criteria. Selected miRNAs were validated by end-point PCR and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), confirmed the existence of conserved miRNAs in hop. Based on the characteristic that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences, a total of 47 potential miRNA targets were identified in hop. Strikingly, the majority of predicted targets were belong to transcriptional factors which could regulate hop growth and development, including leaf, root and even cone development. Moreover, the identified miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and other physiological processes. The cis-regulatory elements relevant to biotic and abiotic stress, plant hormone response, flavonoid biosynthesis were identified in the promoter regions of those miRNA genes. Overall, findings from this study will accelerate the way for further researches of miRNAs, their functions in hop and shows a path for the prediction and analysis of miRNAs to those species whose genomes are not available.

Expression of SANT/HTH Myb mRNA, a plant morphogenesis-regulating transcription factor, changes due to viroid infection.

Potato spindle tuber viroid (PSTVd) belongs to plant-pathogenic, circular, non-coding RNAs. Its propagation is accompanied by (mis)regulation of host genes and induction of pathogenesis symptoms including changes of leaf morphogenesis depending on the strength of viroid variant. We found strong genotype-dependent suppression of tomato morphogenesis-regulating transcription factor SANT/HTH-Myb (SlMyb) due to viroid pathogenesis. Its relative mRNA level was found to be significantly decreased in PSTVd-sensitive tomato (cvs Rutgers and Heinz 1706) due to degradation processes, but increased in PSTVd-tolerant (cv. Harzfeuer). In heterologous system of Nicotiana benthamiana, we observed a SlMyb-associated necrotic effect in agroinfiltrated leaf sectors during ectopic overexpression. Leaf sector necroses were accompanied by activation of nucleolytic enzymes but were suppressed by a strongly pathogenic PSTVd variant. Contrary to that, PSTVd's effect was inhibited by the silencing suppressor p19. It was found that in both, Solanum lycopersicum leaves and N. benthamiana leaf sectors, SlMyb mRNA degradation was significantly stronger in viroid-infected tissues. Necroses induction as well as gene silencing experiments using the SANT/HTH-Myb homologues revealed involvement of this Myb in physiological changes like distortions in flower morphogenesis and growth suppression.

Capillary electrophoresis in an extended nanospray tip-electrospray as an electrophoretic column.

Capillary electrophoresis coupled to mass spectrometry (CE/MS) is gaining its space among the most powerful tools in modern (bio)analytical laboratory. The most challenging instrumental aspect in CE/MS is striking the balance between the stability and reproducibility of the signal and sensitivity of the analysis. Several interface designs have been published in the past decade addressing the variety of instrumental aspects and ease of operation. Most of the interfaces can be categorized either into the sheath flow arrangement (considered to be a de facto standard), or sheathless interface, often expected to provide the ultimate sensitivity. In this work we have explored an "interface-free" approach, where the CE/MS analysis was performed in narrow bore (<20 µm ID) electrospray capillary. The separation capillary and electrospray tip formed one entity and the high voltage, applied at the injection end of the capillary served for both the separation and electrospray ionization. Thus the separation voltage was defined as the product of the electrospray current and resistivity of the separation electrolyte. Optimum conditions for the separation and electrospray ionization were achieved with voltage programming. The performance of this simplest possible CE/MS system was tested on peptide separations from the cytochrome c tryptic digest. The subnanoliter sample consumption and sensitivity in the attomole range predetermines such a system for analysis of limited samples.