A microchip device to enhance free flow electrophoresis using controllable pinched sample injections

Micro free flow electrophoresis (µFFE) is a valuable technique capable of high throughput rapid microscale electrophoretic separation along with mild operating conditions. However, the stream flow separation nature of free flow electrophoresis affects its separation performance with additional stream broadening due to sample stream deflection. To reduce stream broadening and enhance separation performance of µFFE, we presented a simple microfluidic device that enables injection bandwidth control. A pinched injection was formed in the reported µFFE system using operating buffer at sample flow rate ratio (r) setting. Initial bandwidth at the entrance of separation chamber can be shrunk from 800 to 30 µm when r increased from 1 to 256. Stream broadening at the exit of separation chamber can be reduced by about 96% when r increased from 4 to 128, according to both theoretical and experimental results. Moreover, the separation resolution for a dye mixture was enhanced by a factor of 4 when r increased from 16 to 128, which corresponded to an 80% reduction in sample initial bandwidth. Furthermore, a similar enhancement on amino acids separation was obtained by using injection control in the reported µFFE device and readily integrated into online/offline sample preparation and/or downstream analysis procedures.     

A very thin coating for capillary zone electrophoresis of proteins based on a tri(ethylene glycol)-terminated alkyltrichlorosilane

We describe the use of a tri(ethylene glycol)-terminated alkyltrichlorosilane to create a very thin, protein-resistant "self-assembled monolayer" coating on the inner surface of a fused-silica capillary. The same compound has been demonstrated previously on flat silica substrates to resist adsorption of many proteins. As a covalently bound capillary coating, it displays good resistance to the adsorption of cationic proteins, providing clean separations of a mixture of lysozyme, cytochrome c, ribonuclease A, and myoglobin for more than 200 consecutive runs. Electroosmotic flow (EOF) was measured as a function of pH; the coated capillary retains significant cathodal EOF, with roughly 50% of the EOF of an uncoated capillary at neutral pH, making this coating promising for applications requiring some EOF. The EOF was reasonably stable, with a 2.9% relative standard deviation during a 24 h period consisting of 72 consecutive separations of cationic proteins. Efficiencies for cationic protein separations were moderate, in the range of 190,000-290,000 theoretical plates per meter. The coating procedure was simple, requiring only a standard cleaning procedure followed by a rinse with the silane reagent at room temperature. No buffer additives are required to maintain the stability of the coating, making it flexible for a range of applications, potentially including capillary electrophoresis-mass spectrometry (CE-MS).     

Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)-based microchips

MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 mM sodium tetraborate, 25 mM SDS, and 20% v/v ACN. A separation of BSA using this buffer in a 3.0 cm long channel generated a peak with a plate height of 0.38 microm in <20 s. Additional fast separations of myoglobin, alpha-lactalbumin, lysozyme, and cytochrome c also yielded peaks with plate heights ranging from 0.54 to 0.72 microm. All proteins migrated with respect to their individual pIs. To improve the separations, we used a PDMS serpentine chip with tapered turns and a separation distance of 25 cm. The number of plates generated increased linearly with increasing separation distance on the extended separation channel chips; however, the resolution reached an asymptotic value after about 7 cm. This limited the peak capacity of the separation technique to 10-12.     

Modification of silica surfaces for CZE by adsorption of non-ionic hydrophilic polymers or use of radial electric fields

Polyvinylalcohols (PVA) and hydroxyethylcelluloses (HEC) have been used as additives in cyclodextrin-modified capillary zone electrophoretic chiral separations of aromatic amines such as tocainide and its analogs in unpretreated 50 μm i.d. fused silica capillaries. The additives were used at low concentrations (<0.05%) in common buffers, together with the γ-cyclodextrin as chiral selector. They reduce the electroosmotic flow, i.e. increase the migration times of the analytes in these chiral separations, and, moreover, considerably improve both peak symmetry and the widths of the peaks relative to migration time. In terms of the chromatographic theory of efficiency, more than 200000 theoretical plates can be achieved with unpretreated fused silica capillaries. This enhancement of efficiency arises because adsorptive “dynamic” coating with the hydroxylic modifier molecules suppresses adsorption of the analyte molecules by the capillary walls. The influence of field strength and buffer composition on the separation efficiency attainable with and without modifier in the buffers has also been investigated. Alternative experiments on the influence of analyte adsorption on efficiency have been performed by superimposing radial electric fields on the capillary to modify the ζ potentials. Although the EOF could be freely adjusted, it was not possible to obtain an improvement in efficiency comparable with that furnished by coating the adsorptive surface with PVA or HEC.     

Dynamic coating for resolving rhodamine B adsorption to poly(dimethylsiloxane)/glass hybrid chip with laser-induced fluorescence detection

In this paper a method was described about dynamic coating for resolving rhodamine B (RB) adsorption on a hybrid poly(dimethylsiloxane) (PDMS)/glass chip. The results showed that when the non-ionic surfactant Triton X-100 was higher than 0.5% (v/v) into the phosphate buffer, the adsorption of RB appeared. Besides, some separation conditions for RB were investigated, including concentration of Triton X-100, concentration and pH value of running buffer, separation voltage and detection site. Through comparing electroosmotic flow, plate numbers and other parameters, an acceptable separation condition was obtained. Under optimized conditions, the precisions of RB detection (R.S.D., n = 10) were 2.62% for migration time, 4.78% for peak height respectively. Additionally, RB concentration linearity response was excellent with 0.9996 of correlation coefficient between 1 and 100 μM, and a limit of detection (S/N = 3) was 0.2 μM. Finally, we separated rhodamine B isothiocyanate and lysine deriving from the fluorescent probe, and the result displayed that the dynamic coating method was applicable by CE separations using PDMS/glass chip.     

The high concentration and uniform distribution of diamond particles in Ni‐diamond composite coatings by sediment co‐deposition

Ni‐diamond composite coatings with high concentration and uniform distribution of diamond particles were prepared by using sediment co‐deposition (SCD) technique from Watts‐type electrolyte without any additives. The surface and cross‐section morphology was evaluated by optical microscope (OM) and scanning electron microscopy (SEM). It was demonstrated that the Ni‐monolayer diamond composite coatings ~40 ± 5 µm was successfully prepared by the new developed setup for SCD technique. Using this new developed setup, high concentration and uniform distribution of diamond particles of Ni‐monolayer diamond composite coatings were easily fabricated. The wear resistance and cutting performance of obtained composite coatings were also investigated. The results revealed that anti‐wear and cutting performance is superior to those prepared via conventional co‐electrodeposition (CED) technique and pure Ni coatings. In the SCD process, with the increasing diamond content, the wear resistance is approximately the same, and the cutting performance decreases. Therefore, not only the diamond particle content is responsible for the wear resistance and cutting performance, the distribution of diamond particles is also very important factor. Copyright © 2014 John Wiley & Sons, Ltd.     

A cationic β‐cyclodextrin as a dynamic coating for the separation of proteins in capillary electrophoresis

A cationic cyclodextrin was used as dynamic coating for the capillary electrophoresis of a model mixture of proteins (i.e., ubiquitin, α‐lactoglobulin, cytochrome‐c, and myoglobin) as positively charged species in a fused silica capillary. An interesting feature of the coating is that by simple adjustment of the concentration of cyclodextrin added into the background electrolyte, a neutral or positively charged surface, which was beneficial in preventing protein adsorption at the inner capillary wall surface, was obtained. This is the first demonstration of a dynamic coating that yielded a neutral surface for protein separations in capillary electrophoresis. Based on electro‐osmotic flow measurements, addition of 0.05 to 0.10 mg/mL quaternary β‐cyclodextrin in a low pH electrolyte resulted in a neutral or positive surface (undetectable to very slow anodic electro‐osmotic flow). The coating approach afforded the electrophoretic separation of the mixture of proteins at positive polarity with good repeatability and separation performance.     

Electrophoretic separations of neuromediators on microfluidic devices

In the present work, on-chip capillary electrophoresis for the separation of neuromediators is demonstrated. The influence of separation buffer (composition, pH, SDS additive), on-chip electrokinetic sample stacking, and surface pretreatment of the PDMS-PDMS and hybrid PDMS-glass devices on the electrokinetic characteristics of microfluidics (ν_eo, μ_eo, ζ) and separation performance of on-chip capillary electrophoresis of neuromediators have been investigated. It is demonstrated that for the effective separation of neuropeptides on elastomer-based microfluidic devices, on-chip sample stacking is necessary. Field-amplified sample stacking for electroosmotic flow supported on-chip separations of neuromediators and without special design of the sample injection scheme has been demonstrated. Electrophoretic separations of fluorescently labeled analytes have been achieved within tens of seconds at injection volumes of about 110 pL, with plate numbers varying from <1000 to ∼22,000. These results demonstrate that on-chip separation methods with hybrid PDMS-glass devices are perspective for the analysis of (neuro)peptides in small volumes.     

Microcontact printing of multiproteins on the modified mica substrate and study of immunoassays

Microcontact printing (µCP) is an easy and efficient way of producing patterns of self‐assembled monolayers (SAM) containing different functional groups. We have developed a simple and convenient µCP‐based technique for the modification of a mica substrate with 3‐aminopropyltriethoxysilane (APTES) and the micropatterning of proteins (chicken IgG and rabbit IgG) on the modified mica surface. Our approach provides a quick and easy way to produce protein patterns on solid surfaces. The printed immunoglobulin patterns were detected by exposing the substrate to solutions containing fluorescently labeled complementary anti‐IgGs, and the formed immunoassays were studied using fluorescence microscopy. Copyright © 2005 John Wiley & Sons, Ltd.     

Electroosmotic flow in fused deposition modeling (FDM) 3D-printed microchannels

Electroosmotic flow (EOF) was determined in tridimensional (3D)-printed microchannels with dimensions smaller than 100 µm. Fused deposition modeling 3D printing using thermoplastic filaments of PETG (polyethylene terephthalate glycol), PLA (polylactic acid), and ABS (acrylonitrile butadiene styrene) were used to fabricate the microchannels. The current monitoring method and sodium phosphate solutions at different pH values (3–10) were used for the EOF mobility (µ_EOF) measurements, which ranged from 2.00 × 10⁻⁴ to 12.52 × 10⁻⁴ cm² V⁻¹ s⁻¹. The highest and the smallest µ_EOF were obtained for the PLA and PETG microchannels, respectively. Adding the cationic surfactant cetyltrimethylammonium bromide to the sodium phosphate solution caused EOF direction reversion in all the studied microchannels. The obtained results can be interesting for developing 3D-printed microfluidic devices, in which EOF is relevant.     

Cationic poly(amidoamine) dendrimers as additives for capillary electroseparation and detection of proteins

We examine the influence of cationic poly(amidoamine) (PAMAM) dendrimers on capillary electroseparation–UV analysis of proteins. PAMAMs adsorbing to the capillary surface suppressed the wall‐adsorption of proteins; meanwhile, PAMAMs added to the buffer exhibited selectivity toward proteins. Presence of 3×10^?4 g/mL PAMAM generation one (G 1.0) in 30 mM phosphate, at pH 2.6, rendered significant enhancement in separation efficiency; the merged peaks of myoglobin and trypsin inhibitor were separated. Moreover, the protein–dendrimer interactions changed the inherent UV absorbance profiles of proteins. UV–Vis study showed that the absorbance of cytochrome C and transferrin increased at the detection wavelength of 214 nm; their detection sensitivity enhanced by 2.44 and 2.01‐folds, respectively, with addition of 5×10^?4 g/mL PAMAM G 1.0.     

Amperometric Magnetoimmunosensors for Direct Determination of D‐Dimer in Human Serum

Two different D‐dimer disposable amperometric immunosensing designs based on indirect competitive or sandwich formats and the use of carboxylic acid‐modified magnetic beads (COOH‐MBs) and screen‐printed carbon electrodes (SPCEs) have been developed and compared. In both approaches, the resulting modified MBs were magnetically captured on the surface of a SPCE which was used as the transducer for the electrochemical detection at ?0.20 V upon addition of H₂O₂, and hydroquinone (HQ). Both configurations exhibited linear ranges of clinical usefulness and detection limits quite below the clinical threshold (0.5 µg mL^?1 D‐dimer). The sandwich configuration has been successfully tested with serum samples.     

Fracture resistance measurement of fused deposition modeling 3D printed polymers

A method is presented to characterize the fracture resistance and interlayer adhesion of fused deposition modeling (FDM) 3D printed materials. Double cantilever beam (DCB) specimens of acrylonitrile butadiene styrene (ABS) were designed and printed with a precrack at the layers' interface. The DCBs were loaded in an opening mode and the load-displacement curves were synchronized with the optical visualization of the crack tip to detect the critical load at the crack initiation. A finite element model, coupled with J-integral method and fracture surface analysis was then developed to obtain the apparent fracture resistance (J_cr,a) and the interlayer fracture resistance (J_cr,i), as a measure of the interlayer adhesion. The maximum J_cr,i was measured to be 4017 J/m², a value close to the fracture resistance of bulk ABS. Both J_cr,a and J_cr,i increased with the printing temperature. This method can find a great importance in the structural applications of printed materials.     

On-column silver substrate synthesis and surface-enhanced Raman detection in capillary electrophoresis

A new, simple, and efficient approach for on-column surface-enhanced Raman scattering (SERS) detection in capillary electrophoresis (CE) is reported. A ∼50-μm SERS substrate spot was prepared by laser-induced growth of silver particles in the 100-μm inner diameter CE capillary window or in a flow cell consisting of a 250-μm inner diameter fused silica capillary connector. For this purpose, the Raman laser was focused by a 20× objective into the detection window filled with a 0.5 mM silver nitrate and 10 mM citrate buffer solution. During the CE runs, the silver substrate spot was formed in a few seconds after the analyte injection, hence the analytes adsorbed sequentially to the silver surface when the detection window was reached, followed by desorption from the silver surface and continuing the electrophoretic migration to the capillary end. Thus, beyond migration time, valuable molecular specific information was delivered by the SERS spectra. Accurate separations and high-intensity SERS spectra are shown by CE-SERS time-dependent 3D electropherograms for the analytes rhodamine 6G, 4-(2-pyridylazo)resorcinol (PAR), PAR complex with Cu(II) and methylene blue at 0.25–25 ppm concentrations, by using 1.4–3.6 mW HeNe laser power and an acquisition time of 5 s for each spectrum. Before and after each analyte passes the detection window, clean background spectra were recorded and no memory effects perturbed the SERS detection. The silver substrate is characterized by a fast preparation rate, good reproducibility, a preparation success rate of over 95% and no mentionable influence on the electrophoretic migration time, the CE-SERS and CE-UV electropherograms being in good agreement. The successful coupling of CE and on-column SERS detection opens new perspectives for monitoring CE separations.     

Adsorption and adhesion studies of PdSn‐nanoparticles on protonated amine and carboxylic acid‐terminated surfaces

Electroless plating of acrylonitrile‐butadiene‐styrene‐terpolymers (ABS‐plastics) is used for decorative applications and relies on the immobilization of catalytic palladium‐tin nanoparticles. We used chemical force microscopy to measure the adhesion force of palladium‐tin nanoparticles on a patterned amine and carboxyl‐terminated surface prepared by micro‐contact printing. The kinetics of the adsorption process and the population density of the nanoparticles on amine and carboxyl‐terminated surfaces were monitored by quartz crystal microbalance with dissipation analysis. The surface chemistry was investigated by means of polarization‐modulated infrared reflection absorption spectroscopy and X‐ray photoelectron spectroscopy. Enhanced adhesion and population density of PdSn nanoparticles on protonated amine‐terminated surfaces compared with carboxyl‐terminated surfaces is observed. Copyright © 2016 John Wiley & Sons, Ltd.     

Optimisation of the plasma electrolytic oxidation process efficiency on aluminium

Plasma electrolytic oxidation (PEO) is a surface treatment technology enabling fabrication of adherent thick (50–150 µm) coatings on light metal alloys with significantly enhanced hardness, wear and corrosion resistance compared with other conventional treatments. The technology has the potential to play a significant role in the transport sector for replacement of steel with light‐weight materials of improved durability. A main limitation of PEO lies in its relatively high cost, associated with high energy consumption and low coating efficiency. The present work explores possible routes to improve the process efficiency. It is shown that a combination of conventional pre‐anodising with sequential PEO treatment reduces specific energy consumption up to five times because of an increase of the coating growth rate, up to 10 µm/min, compared with existing PEO processes. A further approach to improved coating efficiency involves PEO in electrolytes with suspended fine or nanoparticles, which results in the formation of thicker coatings in reduced time as a result of the incorporation of the particles from the electrolyte into the coating. Additionally, melting of the coating material during the micro‐arc discharge process leads to formation of stabilised high‐temperature phases, such as tetragonal and cubic zirconia, which provide significantly improved microhardness of the coating material and give a potential for thermal barrier applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of an open-tubular trypsin reactor for on-line digestion of proteins

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm⁻² trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol μL⁻¹ horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.     

Open-tubular nanoelectrochromatography (OT-NEC): gel-free separation of single stranded DNAs (ssDNAs) in thermoplastic nanochannels

Electrophoresis or electrochromatography carried out in nanometer columns (width and depth) offers some attractive benefits compared to microscale columns. These advantages include unique separation mechanisms that are scale dependent, fast separation times, and simpler workflow due to the lack of a need for column packing and/or wall coatings to create a stationary phase. We report the use of thermoplastics, in this case PMMA, as the substrate for separating single-stranded DNAs (ssDNAs). Electrophoresis nanochannels were created in PMMA using nanoimprint lithography (NIL), which can produce devices at lower cost and in a higher production mode compared to the fabrication techniques required for glass devices. The nanochannel column in PMMA was successful in separating ssDNAs in free solution that was not possible using microchip electrophoresis in PMMA. The separation could be performed in <1 s with resolution >1.5 when carried out using at an electric field strength of 280 V/cm and an effective column length of 60 μm (100 nm × 100 nm, depth and width). The ssDNAs transport through the PMMA column was driven electrokinetically under the influence of an EOF. The results indicated that the separation was dominated by chromatographic effects using an open tubular nano-electrochromatography (OT-NEC) mode of separation. Interesting to these separations was that no column packing was required nor a wall coating to create the stationary phase; the separation was affected using the native polymer that was UV/O₃ activated and an aqueous buffer mobile phase.     

Design and optimization of a fused‐silica microfluidic device for separation of trivalent lanthanides by isotachophoresis

Elemental analysis of rare earth elements is essential in a variety of fields including environmental monitoring and nuclear safeguards; however, current techniques are often labor intensive, time consuming, and/or costly to perform. The difficulty arises in preparing samples, which requires separating the chemically and physically similar lanthanides. However, by transitioning these separations to the microscale, the speed, cost, and simplicity of sample preparation can be drastically improved. Here, all fourteen non‐radioactive lanthanides (lanthanum through lutetium minus promethium) are separated by ITP for the first time in a serpentine fused‐silica microchannel (70 µm wide × 70 µm tall × 33 cm long) in <10 min at voltages ≤8 kV with limits of detection on the order of picomoles. This time includes the 2 min electrokinetic injection time at 2 kV to load sample into the microchannel. The final leading electrolyte consisted of 10 mM ammonium acetate, 7 mM α‐hydroxyisobutyric acid, 1% polyvinylpyrrolidone, and the final terminating electrolyte consisted of 10 mM acetic acid, 7 mM α‐hydroxyisobutyric acid, and 1% polyvinylpyrrolidone. Electrophoretic electrodes are embedded in the microchip reservoirs so that voltages can be quickly applied and switched during operation. The limits of detection are quantified using a commercial capacitively coupled contactless conductivity detector (C⁴D) to calculate ITP zone lengths in combination with ITP theory. Optimization of experimental procedures and reproducibility based on statistical analysis of subsequent experimental results are addressed. Percent error values in band length and conductivity are ≤8.1 and 0.37%, respectively.     

Surface electric field manipulation of the adsorption kinetics and biocatalytic properties of cytochrome c on a 3D macroporous Au electrode

Upon adsorbing on a solid-state substrate, water-soluble proteins are prone to denaturation and deterioration of their functions due to the conformation change. The surface electric field of a conductive substrate is one of the important factors that influence the character of adsorbed proteins. In this work, a 3D macroporous gold electrode has been prepared and served as the working electrode to study the influence of surface electric field on the adsorption kinetics and conformation of the adsorbed cytochrome c (cyt-c) with the help of electrochemical, in situ electrochemical IR spectroscopic, atomic force microscopic, and contact angle measurements. The external electric field creates excess surface charge which can manipulate the adsorption rate of proteins on the substrate by the enhanced electrostatic interactions between the electrode and protein patches by coupling with complementary charges. The amount of immobilized cyt-c with electrochemical activity on the 3D macroporous gold electrode showed a minimum at potential of zero charge (PZC) and it increased with increasing net excess surface charge. Higher electric field could influence the conformation and the corresponding properties such as direct electrochemistry, bioactivity, and surface character of the adsorbed cyt-c molecules. However, high external electric field leads to damage of the protein secondary structure. This study provides fundamentals for the fabrication of biomolecular devices, biosensors, and biofuel cells through electrostatic interactions. Figure Two cases are illustrated for the protein immobilized on electrode surfaces: a retention of protein structure under moderate excess surface charge, b denaturation and conformation change of proteins adsorbed at high excess surface charge, e.g., due to the higher external electric field.