Calibration-free concentration determination of charged colloidal nanoparticles and determination of effective charges by capillary isotachophoresis

Although colloidal nanoparticles show an electrophoretic heterogeneity under the conditions of capillary electrophoresis, which can be either due to the particle-size distribution and/or the particle shape distribution and/or the zeta-potential distribution, they can form correct isotachophoretic zones with sharp-moving boundaries. Therefore, the technique of isotachophoresis permits to generate plugs in which the co-ions and counter ions of the original colloidal solution are removed and replaced by the buffering counter ions of the leading electrolyte. It is shown that analytical isotachophoresis can be used to measure directly, without calibration, the molar (particle) concentration of dispersed ionic colloids provided that the transference number and the mean effective charge number of the particles (within the isotachophoretic zone) can be determined with adequate accuracy. The method can also be used to measure directly the effective charge number of biomacromolecules or colloidal particles, if solutions with known molar (particle) concentration can be prepared. The validity of the approach was confirmed for a model solution containing a known molar concentration of bovine serum albumin.      

Automated extraction of direct,reactive, and vat dyes from cellulosic fibers for forensic analysis by capillary electrophoresis

Systematic designed experiments were employed to find the optimum conditions for extraction of direct, reactive, and vat dyes from cotton fibers prior to forensic characterization. Automated microextractions were coupled with measurements of extraction efficiencies on a microplate reader UV–visible spectrophotometer to enable rapid screening of extraction efficiency as a function of solvent composition. Solvent extraction conditions were also developed to be compatible with subsequent forensic characterization of extracted dyes by capillary electrophoresis with UV–visible diode array detection. The capillary electrophoresis electrolyte successfully used in this work consists of 5 mM ammonium acetate in 40:60 acetonitrile–water at pH 9.3, with the addition of sodium dithionite reducing agent to facilitate analysis of vat dyes. The ultimate goal of these research efforts is enhanced discrimination of trace fiber evidence by analysis of extracted dyes. Figure Fitted absorbance response surface for extraction of a direct dye, C. I. yellow 58, using a ternary solvent system.     

Two-dimensional electrophoresis on a microfluidic chip for quantitative amino acid analysis

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated. Figure       

Patterning of 293T fibroblasts on a mica surface

Controllable cell growth on the defined areas of surfaces is important for potential applications in biosensor fabrication and tissue engineering. In this study, controllable cell growth was achieved by culturing 293 T fibroblast cells on a mica surface which had been patterned with collagen strips by a microcontact printing (μCP) technique. The collagen area was designed to support cell adhesion and the native mica surface was designed to repel cell adhesion. Consequently, the resulting cell patterns should follow the micro-patterns of the collagen. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) measurement, atomic-force microscope (AFM) observation, and force-curve measurement were used to monitor property changes before and after the collagen adsorption process. Further data showed that the patterned cells were of good viability and able to perform a gene-transfection experiment in vitro. This technique should be of potential applications in the fields of biosensor fabrication and tissue engineering. Figure Controllable cells growth has been achieved by culturing 293T fibroblast cells on the mica surface which had been patterned with collagen strips by microcontact printing (μCP) technique     

Optical sensing in microfluidic lab-on-a-chip by femtosecond-laser-written waveguides

We use direct femtosecond laser writing to integrate optical waveguides into a commercial fused silica capillary electrophoresis chip. High-quality waveguides crossing the microfluidic channels are fabricated and used to optically address, with high spatial selectivity, their content. Fluorescence from the optically excited volume is efficiently collected at a 90° angle by a high numerical aperture fiber, resulting in a highly compact and portable device. To test the platform we performed electrophoresis and detection of a 23-mer oligonucleotide plug. Our approach is quite powerful because it allows the integration of photonic functionalities, by simple post-processing, into commercial LOCs fabricated with standard techniques. Figure Femtosecond laser written waveguides can selectively excite fluorescence in a microfluidic channel of a commercial lab-on-a-chip. A compact scheme for on-chip detection by laser induced fluorescence is applied to capillary electrophoresis of a 23-mer Cy3-labeled oligonucleotide     

Surface-enhanced Raman scattering for protein detection

Proteins are essential components of organisms and they participate in every process within cells. The key characteristic of proteins that allows their diverse functions is their ability to bind other molecules specifically and tightly. With the development of proteomics, exploring high-efficiency detection methods for large-scale proteins is increasingly important. In recent years, rapid development of surface-enhanced Raman scattering (SERS)-based biosensors leads to the SERS realm of applications from chemical analysis to nanostructure characterization and biomedical applications. For proteins, early studies focused on investigating SERS spectra of individual proteins, and the successful design of nanoparticle probes has promoted great progress of SERS-based immunoassays. In this review we outline the development of SERS-based methods for proteins with particular focus on our proposed protein-mediated SERS-active substrates and their applications in label-free and Raman dye-labeled protein detection. Figure Protein-mediated SERS-active substrates for protein detection     

Nuclear magnetic resonance of mass-limited samples using small RF coils

Figure Schematic diagram of a typical arrangement used for hyphenating chemical microseparations (e.g. capillary HPLC, CE, or CEC) with microcoil NMR detection     

Capillary electrophoretic separation of humic substances using hydroxyethyl cellulose as a buffer additive and its application to characterization of humic substances in a river water sample

We have developed a concise tool for the investigation of the transition of humic substances in environmental water. The separation of water-soluble humic substances was achieved rapidly and effectively by capillary electrophoresis using a polyacrylamide-coated capillary and a phosphate electrophoretic buffer solution (pH 7.0) containing hydroxyethyl cellulose. The separation mechanism was assessed using the ultrafiltration technique. The effect of the complexation of humic substances with metal ions was studied by using the proposed method. When Fe(III) ions or EDTA was added to the sample solution of fulvic acid, a distinct change in the electropherogram pattern based on the conformational change of fulvic acid was observed. The successful application of the proposed method to the characterization of humic substances in a river water sample was also demonstrated. Figure Addition of Fe(III) ions or EDTA to a solution containing fulvic acid (FA) results in a distinct change in the electropherogram pattern, which reflects the conformational change of FA: this forms the basis for the characterization of humic substances in river water samples     

Critical evaluation of sample pretreatment techniques

Sample preparation before chromatographic separation is the most time-consuming and error-prone part of the analytical procedure. Therefore, selecting and optimizing an appropriate sample preparation scheme is a key factor in the final success of the analysis, and the judicious choice of an appropriate procedure greatly influences the reliability and accuracy of a given analysis. The main objective of this review is to critically evaluate the applicability, disadvantages, and advantages of various sample preparation techniques. Particular emphasis is placed on extraction techniques suitable for both liquid and solid samples. Figure Miniaturised extraction techniques allow sensitive analysis of also small sample volumes.     

Application of surface chemical analysis tools for characterization of nanoparticles

The important role that surface chemical analysis methods can and should play in the characterization of nanoparticles is described. The types of information that can be obtained from analysis of nanoparticles using Auger electron spectroscopy (AES), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (TOF-SIMS), low-energy ion scattering (LEIS), and scanning-probe microscopy (SPM), including scanning tunneling microscopy (STM) and atomic force microscopy (AFM), are briefly summarized. Examples describing the characterization of engineered nanoparticles are provided. Specific analysis considerations and issues associated with using surface-analysis methods for the characterization of nanoparticles are discussed and summarized, with the impact that shape instability, environmentally induced changes, deliberate and accidental coating, etc., have on nanoparticle properties.      

Rapid analysis of native neomycin components on a portable capillary electrophoresis system with potential gradient detection

A simple method based on capillary electrophoresis with potential gradient detection was developed to separate and detect neomycin components within 4 min without a derivatization step. Satisfactory separation and good repeatability were obtained using a separation buffer composed of 1 mM ammonium citrate (pH 3.5). The linearity of the method ranged from 10 to 1000 ppm with a limit of detection for neomycin B of about 7 ppm. After a simple dilution and filtering pretreatment step, neomycin components in three real samples were successfully analyzed without any major interference. Due to its simplicity and reliability, this method could provide an excellent alternative to the assays currently listed in U.S. and European Pharmacopoeia. The experiments were performed on a portable capillary electrophoresis system and, hence, the method can be readily applied to field analysis and point-of-care testing. Figure Photo of portable CE-P2-PGD system     

On-column labeling for capillary electrophoretic analysis of individual mitochondria directly sampled from tissue cross sections

This technical note reports on a new procedure to on-column-label organelles sampled from a tissue cross section into a fused silica capillary. These organelles are then analyzed by capillary electrophoresis with postcolumn laser-induced fluorescence detection. In this procedure, the fluorescent label does not come in contact with the tissue, which facilitates visualization of the sampled tissue cross section. In addition, on-column labeling allows for better control of the reaction time and fluorescent label concentrations. As a proof-of-principle, we show results of mitochondria from rat gastrocnemius muscle cross sections that were on-column-labeled with 10-N-nonyl acridine orange (NAO), a mitochondrion-specific probe, and compare them with results for NAO in-tissue labeling of the same tissue. The new organelle labeling procedure reported here may easily be extended to the analysis of individual organelles in other biological samples and may become a valuable tool in studies investigating the role of mitochondria in muscle aging and exercise physiology.      

Ultrafast plasmon dynamics and evanescent field distribution of reproducible surface-enhanced Raman-scattering substrates

Surface-enhanced Raman scattering (SERS) is a potent tool in bioanalytical science because the technique combines high sensitivity with molecular specificity. However, the widespread and routine use of SERS in quantitative biomedical diagnostics is limited by tight requirements on the reproducibility of the noble metal substrates used. To solve this problem, we recently introduced a novel approach to reproducible SERS substrates. In this contribution, we apply ultrafast time-resolved spectroscopy to investigate the photo-induced collective charge-carrier dynamics in such substrates, which represents the fundamental origin of the SERS mechanism. The ultrafast experiments are accompanied by scanning-near field optical microscopy and SERS experiments to correlate the appearance of plasmon dynamics with the resultant evanescent field distribution and the analytically relevant SERS enhancement. Figure Ultrafast time-resolved differential absorption spectroscopy combined with scanning near-field optical microscopy (left) and atomic force microscopy (right) yields insight into the photoinduced charge-carrier dynamics in innovative reproducible SERS-substrates Dana Cialla and Ronald Siebert contributed equally to this work.     

Forensic analysis of anthraquinone,azo, and metal complex acid dyes from nylon fibers by micro-extraction and capillary electrophoresis

The extraction and separation of dyes present on textile fibers offers the possibility of enhanced discrimination between forensic trace fiber evidence. An automated liquid sample handling workstation was programmed to deliver varying solvent combinations to acid-dyed nylon samples, and the resulting extracts were analyzed by an ultraviolet/visible microplate reader to evaluate extraction efficiencies at different experimental conditions. Combinatorial experiments using three-component mixture designs varied three solvents (water, pyridine, and aqueous ammonia) and were employed at different extraction temperatures for various extraction durations. The extraction efficiency as a function of the three solvents (pyridine/ammonia/water) was modeled and used to define optimum conditions for the extraction of three subclasses of acid dyes (anthraquinone, azo, and metal complex) from nylon fibers. The capillary electrophoresis analysis of acid dye extracts is demonstrated using an electrolyte solution of 15 mM ammonium acetate in acetonitrile/water (40:60, v/v) at pH 9.3. Excellent separations and discriminating diode array spectra are obtained even for dyes of similar color. Figure Capillary electropherogram of three acid dyes extracted from nylon 6,6 thread     

Sensitivity improvement in capillary electrophoresis using organo-aqueous separation buffers and thermal lens detection

It is shown that organo-aqueous separation buffers show much promise when used in capillary electrophoresis separations with photothermal (thermal lens) detection systems. Acetonitrile–water and methanol–water mixtures were selected, as conventionally used in capillary electrophoresis. It is shown that, despite more sophisticated experimental conditions (significant heat outflow from the capillary body) and peak detection, the theoretical ratio of the thermal lens signal for a binary mixture to the thermal lens signal for an aqueous solution (or the corresponding ratio obtained experimentally under bulk batch conditions) can be used to predict the sensitivity of thermal lens detection in capillary electrophoresis. The limits of detection for 2-, 3-, and 4-nitrophenols selected as model compounds in 70% v/v acetonitrile separation buffers are 1×10⁻⁶ M, 1×10⁻⁶ M and 3×10⁻⁷ M, respectively, and are therefore decreased by a factor of six compared to thermal lens detection in aqueous separation buffers. The overall increase in the thermal lens detection sensitivity in a 100% ACN buffer is a factor of 13.      

A novel approach for the detection of DNA using immobilized peptide nucleic acid (PNA) probes and signal enhancement by real-time immuno-polymerase chain reaction (RT-iPCR)

A new approach for the detection of DNA target molecules is described, using capture probes and subsequent signal enhancement by a uniform polymerase chain reaction (PCR). Peptide nucleic acid probes were immobilized in real-time PCR-compatible microtiter plates. After hybridization of biotinylated DNA targets, detection was performed by real-time immuno-PCR, a method formerly used for protein detection. We demonstrate the feasibility of this strategy for the qualitative detection of DNA oligonucleotides with a detection limit (LOD) of 6 attomol. Furthermore, the method was applied to PCR-amplified samples from genetically modified maize DNA (Mon810). A 483-bp DNA fragment was detected in mixture with 99.9% of noncomplementary DNA with a sensitivity down to the level of attomole. Figure       

Capillary HPLC–ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water-soluble protein fraction of selenized yeast

A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram level. The method, assisted by information obtained by MALDI TOF MS on the 5000 Da cut-off fraction, permitted the purity of bands and spots to be estimated and the efficiency of tryptic digestion and the quantity of selenium present in individual peptides to be evaluated. Owing to the high sensitivity and the lack of matrix suppression effects, the method provided chromatograms with signal-to-noise ratios of 10–1000 in conditions where the common ES Q–TOF MS detection failed.      

TOF-S-SIMS molecular depth profiling of organic bilayers using mechanical wear test methodology

Recent publications on static secondary ion mass spectrometry (S-SIMS) focus on molecular depth profiling by using polyatomic or ultra-low energy monoatomic projectiles. Since their applicability depends on the relationship between the ion yield and the depth, which is hard to obtain without extensive studies, a combination of a wear test method with S-SIMS surface analysis was performed in the current study. Using this non-sputtering procedure, the relation between the signal intensity and the local concentration remains in principle the same as that at the surface (which is easy to determine). Mechanical erosion was successfully applied to expose sub-surface material from organic multilayers. Through surface analysis with S-SIMS on the gradually exposed deeper planes, molecular depth profiles could be obtained. The study was conducted on a model system relevant to offset printing, consisting of two polymer layers, containing dyes and a surfactant, cast on an Al substrate. Figure Concept of mechanical erosion followed by S-SIMS surface analysis to obtain molecular depth profiles     

Application of XPS and ToF-SIMS for surface chemical analysis of DNA microarrays and their substrates

The chemical composition of the functional surfaces of substrates used for microarrays is one of the important parameters that determine the quality of a microarray experiment. In addition to the commonly used contact angle measurements to determine the wettability of functionalized supports, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) are more specific methods to elucidate details about the chemical surface constitution. XPS yields information about the atomic composition of the surface, whereas from ToF-SIMS, information on the molecular species on the surface can be concluded. Applied on printed DNA microarrays, both techniques provide impressive chemical images down to the micrometer scale and can be utilized for label-free spot detection and characterization. Detailed information about the chemical constitution of single spots of microarrays can be obtained by high-resolution XPS imaging. Figure Eye-catching image for the graphical online abstract     

Fast preparation of polystyrene-based monolith using microwave irradiation for micro-column separation

The conventional analytical HPLC was successfully developed for micro-column separation by using a simple eluate splitting system, a self-preparation of monolithic column and an on-capillary column detector in our laboratory. A typical polystyrene-based monolith was quickly prepared inside the fused-silica capillary, which in situ polymerization was carried out in 10 min by microwave irradiation. The reactant solution consisted of styrene (ST) as a functional monomer, divinylbenzene (DVB) as a cross-linking agent, toluene and isooctane as porogenic solvents, and azobisisobutyronitrile (AIBN) as an initiator. The monolith was proved to form in the center of the capillary and adhered to the column inner wall by the scanning electron micrograph. Its chromatographic behaviors were evaluated in detail by varying the flow rate and percentage of mobile phases, and under the optimal condition, baseline separation of the model analytes including thiourea, benzene, toluene, ethylbenzene was obtained with a highest theoretical plate number near 11,290 N/m by the developed capillary HPLC. Furthermore, the stability and porosity of the prepared monolith were systematically investigated by a simple flow method. Figure A polystyrene-based monolith was rapidly prepared inside the fused-silica capillary, which in situ polymerization was carried out about 10 min by microwave irradiation.