Development and optimization of an integrated PDMS based‐microdialysis microchip electrophoresis device with on‐chip derivatization for continuous monitoring of primary amines

An all‐PDMS on‐line microdialysis‐microchip electrophoresis with on‐chip derivatization and electrophoretic separation for near real‐time monitoring of primary amine‐containing analytes is described. Each part of the chip was optimized separately, and the effect of each of the components on temporal resolution, lag time, and separation efficiency of the device was determined. Aspartate and glutamate were employed as test analytes. Derivatization was accomplished with naphthalene‐2,3,‐dicarboxyaldehyde/cyanide (NDA/CN^?), and the separation was performed using a 15‐cm serpentine channel. The analytes were detected using LIF detection.     

Quantification of D‐Asp and D‐Glu in rat brain and human cerebrospinal fluid by microchip electrophoresis

A microchip electrophoresis (MCE) method with LIF detection was presented for quantification of D ‐aspartic acid (D ‐Asp) and D ‐glutamate (D ‐Glu) in biological samples. D ‐Asp and D ‐Glu were determined after precolumn derivatization with FITC. The chiral separation was performed on a glass/PDMS hybrid microfluidic chip using γ‐CD as chiral selector in the running buffer. High sensitive detection was obtained by the LIF detection. The LODs (S/N = 3) for D ‐Asp and D ‐Glu were 6.0×10^–8 and 4.0×10^–8 M, respectively. Using this method, the levels of D ‐Asp and D ‐Glu in rat brain and human cerebrospinal fluid (CSF) were determined.     

A G‐quadruplex/hemin DNAzyme‐based microchip electrophoresis chemiluminescence assay for highly sensitive detection of biotin in flour

Quantitative analysis of biotin in biological fluids, foods, and pharmaceutical is important for diagnosis and treatment of biotin‐related diseases and health maintenance. In this work, a novel G‐quadruplex/hemin DNAzyme‐based microchip electrophoresis chemiluminescence (CL) assay method was established for rapid and highly sensitive detection of biotin. This method is based on the specific binding between biotin and streptavidin, the catalytic CL characteristics of G‐quadruplex/hemin DNAzyme to the oxidation–reduction reaction of hydrogen peroxide with luminol, and the on‐line separation function of microchip electrophoresis. Under the optimal experimental conditions, on‐chip biotin analysis was achieved within 1 min. The CL intensity is linearly proportional to the concentration of biotin in the range of 13–630 nM with a detection limit of 6.4 nM. The proposed method has been applied for the detection of biotin in flour, biotin contents in three flour samples are found in the range of 199–223 ng/g with a mean value of 214 ng/g. The recoveries were in the range of 94–103%. With excellent sensitivity and good selectivity, the proposed method could be applied in a wide range of biological fluids, foods, and pharmaceutical analysis.     

Rapid quantification of quinine by multi‐stacking in a portable microchip electrophoresis system

A new multi‐stacking pre‐concentration procedure based on field‐enhanced sample injection (FESI), field‐amplified sample stacking, and transient isotachophoresis was developed and implemented in a compact microchip electrophoresis (MCE) with a double T‐junction glass chip, coupled with an on‐chip capacitively coupled contactless conductivity detection (C⁴D) system. A mixture of the cationic target analyte and the terminating electrolyte (TE) from the two sample reservoirs was injected under FESI conditions within the two sample‐loading channels. At the double T‐junction, the stacked analyte zones were further concentrated under field‐amplified stacking conditions and then subsequently focused by transient‐isotachophoresis and separated along the separation channels. The proposed multi‐stacking strategy was verified under a Universal Serial Bus (USB) fluorescence microscope employing Rhodamine 6G as the model analyte. This developed approach was subsequently used to monitor the target quinine present in human plasma samples. The total analysis time for quinine was approximately 200 s with a sensitivity enhancement factor of approximately 61 when compared to the typical gated injection. The detection and quantification limits of the developed approach for quinine were 3.0 μg/mL and 10 μg/mL, respectively, with intraday and interday repeatability (%RSDs, n = 5) of 3.6 and 4.4%. Recoveries in spiked human plasma were 98.1–99.8%.     

Precise determination of N‐acetylcysteine in pharmaceuticals by microchip electrophoresis

A novel microchip electrophoresis method for the rapid and high‐precision determination of N‐acetylcysteine, a pharmaceutically active ingredient, in mucolytics has been developed. Isotachophoresis separations were carried out at pH 6.0 on a microchip with conductivity detection. The methods of external calibration and internal standard were used to evaluate the results. The internal standard method effectively eliminated variations in various working parameters, mainly run‐to‐run fluctuations of an injected volume. The repeatability and accuracy of N‐acetylcysteine determination in all mucolytic preparations tested (Solmucol 90 and 200, and ACC Long 600) were more than satisfactory with the relative standard deviation and relative error values <0.7 and <1.9%, respectively. A recovery range of 99–101% of N‐acetylcysteine in the analyzed pharmaceuticals predetermines the proposed method for accurate analysis as well. This work, in general, indicates analytical possibilities of microchip isotachophoresis for the quantitative analysis of simplified samples such as pharmaceuticals that contain the analyte(s) at relatively high concentrations.     

Integration of continuous-flow sampling with microchip electrophoresis using poly(dimethylsiloxane)-based valves in a reversibly sealed device

Here we describe a reversibly sealed microchip device that incorporates poly(dimethylsiloxane) (PDMS)-based valves for the rapid injection of analytes from a continuously flowing stream into a channel network for analysis with microchip electrophoresis. The microchip was reversibly sealed to a PDMS-coated glass substrate and microbore tubing was used for the introduction of gas and fluids to the microchip device. Two pneumatic valves were incorporated into the design and actuated on the order of hundreds of milliseconds, allowing analyte from a continuously flowing sampling stream to be injected into an electrophoresis separation channel. The device was characterized in terms of the valve actuation time and pushback voltage. It was also found that the addition of sodium dodecyl sulfate (SDS) to the buffer system greatly increased the reproducibility of the injection scheme and enabled the analysis of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde/cyanide. Results from continuous injections of a 0.39 nL fluorescein plug into the optimized system showed that the injection process was reproducible (RSD of 0.7%, n = 10). Studies also showed that the device was capable of monitoring off-chip changes in concentration with a device lag time of 90 s. Finally, the ability of the device to rapidly monitor on-chip concentration changes was demonstrated by continually sampling from an analyte plug that was derivatized upstream from the electrophoresis/continuous flow interface. A reversibly sealed device of this type will be useful for the continuous monitoring and analysis of processes that occur either off-chip (such as microdialysis sampling) or on-chip from other integrated functions.     

Development of a universal serial bus-powered mini-high-voltage power supply for microchip electrophoresis

We describe a miniature high-voltage power supply (HVPS) with dimensions of 4.7 x 5.6 x 2.5 cm (W x L x H) powered by universal serial bus (USB) ports. Two strategies were adopted to ensure its efficient power usage. (i) Only two high-voltage converters (one positive and one negative) and two relays were used for power saving, while keeping the sample plug stable and well-defined and avoiding sample leakage for microchip electrophoresis. (ii) The components and their running modes were specially designed to decrease power waste according to the feature of different periods of the microchip electrophoresis process. Performance of this USB-based mini-HVPS was demonstrated using sodium fluorescein analyte with microchip electrophoresis/LIF detection.     

Protein separation using free‐flow electrophoresis microchip etched in a single step

A one‐step etching method was developed to fabricate glass free‐flow electrophoresis microchips with a rectangle separation microchamber (42 mm‐long, 23 mm‐wide and 28 μm‐deep), in which two glass bridges (0.5 mm‐wide) were made simultaneously to prevent bubbles formed by electrolysis near the Pt electrode from entering the separation chamber. By microchip free‐flow zone electrophoresis, with 200 V voltage applied, the baseline separation of three FITC labeled proteins, ribonuclease B, myoglobin and β‐lactoglobulin, was achieved, with resolution over 1.78. Furthermore, with 2.5 mM Na₂SO₄ added into the electrode buffer to form higher electrical field strength across separation microchamber than electrode compartments, similar resolution of samples was achieved with the applied voltage decreased to 75 V, which could obviously decrease Joule heat during continuous separation. All these results demonstrate that the free‐flow electrophoresis microchip fabricated by one‐step etching method is suitable for the continuous separation of proteins, which might become an effective pre‐fractionation method for proteome study.     

Integration of a carbon microelectrode with a microfabricated palladium decoupler for use in microchip capillary electrophoresis/electrochemistry

A method to integrate a carbon microelectrode with a microfabricated palladium decoupler for use in microchip capillary electrophoresis (CE) is detailed. As opposed to previous studies with decouplers for microchip CE, the working electrode material, which is made by micromolding of a carbon ink, is different from the decoupling electrode material (palladium). The manner in which the working electrode is made does not add additional etching or lithographic steps to the fabrication of the glass electrode plate. The hybrid poly(dimethylsiloxane)/glass device was characterized with fluorescence microscopy and by monitoring the CE-based separation of dopamine. Hydrodynamic voltammograms exhibited diffusion-limited currents occurring at potentials above +1.0 V. It was also shown that the half-wave potential does not shift as the separation potential is changed, as is the case in nondecoupled systems. Gated injections of dopamine in a 25 mM boric acid buffer (pH 9.2) showed a linear response from 200 to 5 microM (r2 = 0.9992), with a sensitivity of 5.47 pA/microM and an estimated limit of detection of 2.3 microM (0.621 fmol, S/N = 3). This is the first report of coupling a carbon electrode with a decoupler in microchip CE.     

Quantitative determination of dopamine in single rat pheochromocytoma cells by microchip electrophoresis with only one high‐voltage power supply

We developed a method for the direct identification of dopamine in single cultured rat pheochromocytoma cells by capillary electrophoresis using an end‐channel carbon fiber nanoelectrode amperometric detector. The operation mode was designed to achieve single‐cell injection and lysis in microfluidic chip electrophoresis with only one high‐voltage power supply. The separation and detection conditions were optimized. Four catecholamines were baseline‐separated and determined with this system, and the cell density and liquid height of the reservoirs were accommodated for single cell loading, docking and analysis. The microchip capillary electrophoresis system was successfully applied to determine dopamine in single cultured rat pheochromocytoma cells.     

Concurrent determination and separation of inorganic cations and anions in microchip electrophoresis with precisely controlled high‐voltage

An improved method for the concurrent determination and separation of cations and anions by microchip electrophoresis with capacitively coupled contactless conductivity detection (ME‐C⁴D) is described. Two kinds of microchip structures were designed. The first microchip has a long bent separation channel. And for the defects of the first microchip, the second microchip with a Y‐type separation channel has been proposed. The background electrolyte (BGE) composed of 20 mm His/MES and 0.01 mm CTAB was optimized for inhibiting the electroosmotic flow (EOF). Due to the low electroosmotic flow, the cations and anions migrate in opposite directions and can be separated from each other. With the precisely controlled high‐voltage, cations and anions can be migrated in microchannels according to our requirements and sequentially detected by a C⁴D detector built in‐house. Samples containing K⁺, Na⁺, Li⁺, Cl⁻, F⁻ and PO₄³⁻ were analyzed simultaneously in a single run (within 140 s) by both methods. The reproducibility obtained by both methods remained below 5% for migration time and within 3.5–9.1% for peak areas. The proposed concurrent determination methods are inexpensive, simple, fast, ease of operation, high degree of integration.     

Organic Spherical Nucleic Acids for the Transport of a NIR‐II‐Emitting Dye Across the Blood–Brain Barrier

DNA nanotechnology plays an increasingly important role in the biomedical field; however, its application in the design of organic nanomaterials is underexplored. Herein, we report the use of DNA nanotechnology to transport a NIR‐II‐emitting nanofluorophore across the blood–brain barrier (BBB), facilitating non‐invasive imaging of brain tumors. Specifically, the DNA block copolymer, PS‐b‐DNA, is synthesized through a solid‐phase click reaction. We demonstrate that its self‐assembled structure shows exceptional cluster effects, among which BBB‐crossing is the most notable. Therefore, PS‐b‐DNA is utilized as an amphiphilic matrix to fabricate a NIR‐II nanofluorephore, which is applied in in vivo bioimaging. Accordingly, the NIR‐II fluorescence signal of the DNA‐based nanofluorophore localized at a glioblastoma is 3.8‐fold higher than the NIR‐II fluorescence signal of the PEG‐based counterpart. The notably increased imaging resolution will significantly benefit the further diagnosis and therapy of brain tumors.     

Determination of resin acid composition in rosin samples using cyclodextrin‐modified capillary electrophoresis

Rosins are used in a wide variety of industries in varnishes, adhesives, drug coatings, etc. In this project a novel capillary electrophoresis method was developed to investigate the resin acid composition of rosins. The acids were separated and the concentrations of individual acids present in gum rosin samples determined in order to investigate any links between the presence and concentration of these acids and the tendency of rosins to crystallize. The capillary electrophoresis method successfully separated nine resin acids in various rosin samples where previously they could not all be separated. Calibration curves were created to determine acid concentration. Abietic, dehydroabietic, neoabietic, pimaric, isopimaric, levopimaric, sandaracopimaric, palustric, and 7‐oxo‐dehydroabietic acids were separated using a 20 mM tris buffer at pH 9 containing 15% methanol 5 mM (2‐hydroxypropyl)‐γ‐cyclodextrin 10 mM sulfobutylether‐β‐cyclodextrin. Their concentrations in a crystallizing and a noncrystallizing rosin sample were determined.     

Development of a potential method based on microchip electrophoresis with fluorescence detection for the sensitive determination of intracellular thiols in RAW264.7 cells

This paper, for the first time, reported the development of a simple, rapid, and reliable method for the separation and sensitive determination of four thiol compounds including homocysteine, cysteine, glutathione, and N‐acetylcysteine based on glass MCE with fluorescence detection using a highly reactive fluorogenic probe, 1,3,5,7‐tetramethyl‐8‐phenyl‐(2‐maleimide)‐difluoroboradiaza‐s‐indacene (TMPAB‐o‐M), as the labeling reagent. TMPAB‐o‐M reacted selectively with thiols to produce highly fluorescent derivatives and the highest derivatization efficiency was achieved within 6 min in physiological conditions. After the optimization of separation conditions, a baseline separation of the four thiol compounds was achieved with the detection limits ranging from 2 nM for glutathione to 4 nM for cysteine (S/N = 3) and RSDs (n = 5) in the range of 3.2–3.8%. The proposed method was significantly sensitive compared to those using electrochemical or even LIF detection in MCE‐based setup reported previously, and applied to the determination of intracellular thiols in macrophage RAW264.7 cells.     

Phosphate‐affinity electrophoresis on a microchip for determination of protein kinase activity

We describe microchip‐based phosphate‐affinity electrophoresis (μPAE) for separation of peptides aimed at determination of kinase activity. The μPAE exploits two recently published technologies: autonomous sample injection for PDMS microchips and a phosphate‐specific affinity ligand, Phos‐tag. We prepared a fluorescently labeled substrate peptide, specific to human c‐Src, and its phosphorylated form. We synthesized a Phos‐tag–poly(dimethylacrylamide) conjugate. The conjugate and the sample solutions were autonomously injected into a PDMS–glass hybrid microchip. The two solutions were contacted together in the microchannel. When the peptides were electrophoresed into the Phos‐tag–poly(dimethylacrylamide) region, the phosphorylated peptide was specifically trapped, and separated from the nonphosphorylated peptide in 10 s. The results were quantified by the areas of the fluorescence peaks. The calibration plot obtained with standard samples showed an excellent linearity and a LOD of 0.9% phosphorylated peptide among the total peptides. For c‐Src‐reacted samples, the results from the μPAE were in good agreement with those from matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The μPAE was also successful in the presence of inhibitors for c‐Src. The measured 50% inhibitory concentration values for staurosporine, PP2, and SU6656 were in good agreement with the literature values.     

Capillary electrophoresis with a UV light-emitting diode source for chemical monitoring of native and derivatized fluorescent compounds

We report the utilization of a high power UV light-emitting diode for fluorescence detection (UV-LED-IF) in CE separations. CE-UV-LED-IF allows analysis of a range of environmentally and biologically important compounds, including PAHs and biogenic amines, including neurotransmitters, amino acids, proteins, and peptides, that have been derivatized with UV-excited fluorogenic labels, e.g., o-phthalic dicarboxaldehyde/beta-mercaptoethanol (OPA/beta-ME). The 365 nm UV-LED was used as a stable, low cost source for detection of UV-excited fluorescent compounds. UV-LED-IF was used with both zonal CE separations and MEKC. Native fluorescence detection of PAHs was accomplished with detection limits ranging from 10 nM to 1.3 microM. Detection limits for OPA/beta-ME-labeled glutamic acid and aspartic acid were 11 and 10 nM, respectively, for off-line labeling, and 47 and 47 nM, respectively, for on-line labeling, comparable to UV-laser-based systems. Analysis of OPA/beta-ME-labeled proteins and peptides was performed with 28 and 47 nM detection limits for BSA and myoglobin, respectively.     

Development of a microchip capillary electrophoresis method for determination of the purity and integrity of mRNA in lipid nanoparticle vaccines

Messenger RNA (mRNA)-based vaccines are advantageous because they can be relatively quicker and more cost efficient to manufacture compared to other traditional vaccine products. Lipid nanoparticles have three common purposes: delivery, self-adjuvanting properties, and mRNA protection. Faster vaccine development requires an efficient and fast assay to monitor mRNA purity and integrity. Microchip CE is known to be a robust technology that is capable of rapid separation. Here, we describe the development and optimization of a purity and integrity assay for mRNA-based vaccines encapsulated in lipid nanoparticles using commercial microchip-based separation. The analytical parameters of the optimized assay were assessed and the method is a stability indicating assay.     

On-line microdialysis sampling coupled with flame atomic absorption spectrometry for continuous in vivo monitoring of diffusible magnesium in the blood of living animals

A novel on-line microdialysis sampling coupled with flame atomic absorption spectrometry (FAAS) with an attractive application is reported. Microdialysates perfused through implanted microdialysis probes were directly introduced into the flame atomizer of a FAAS system using 0.2% HNO₃ as carrier solution at a nebulizer uptake flow rate of 6 ml min⁻¹. The interval for each determination was 90 s (60 s sampling time, 10 s read time and 20 s washing time). The analytical characteristics of the on-line microdialysis-FAAS system were validated as follows: linearity range, 0-300 mg l⁻¹; detection limit (3σ, n = 7), 0.53 mg l⁻¹; precision (R.S.D., n = 50), 4.1%. By comparing Mg levels in the blood of living rabbits with the results obtained from in vivo no net flux (NNF) method, the accuracy of the proposed on-line method was found to be good. The present method can be successfully applied to the in vivo monitoring of diffusible Mg in the blood of living rabbits after magnesium sulfate (MgSO₄) administration with a temporal resolution of 1.5 min.     

Fluorescence‐Labeled Immunomicelles: Preparation,in vivo Biodistribution,and Ability to Cross the Blood–Brain Barrier

Multifunctional hybrid micelles are prepared from amphiphilic mal‐PEG‐b‐PLA and mPEG‐b‐P(LA‐co‐DHC/RhB) block copolymers. A specific anti‐transferrin receptor antibody, OX26, is linked onto the surface of the micelles. ELISA indicates that the conjugated antibody preserves its activity. OX26 conjugation can increase the uptake efficiency of micelles by target cell lines (C6). Pharmacokinetics and in vivo biodistribution experiments are carried out to investigate the ability of OX26‐conjugated micelles (immunomicelles) to cross the blood–brain barrier. The data show that the brain uptake of OX26‐conjugated micelles is much more than that of OX26‐free ones. Therefore, OX26‐conjugated micelles will be promising drug carriers to cross the blood‐brain barrier.

     

Development of an ultra-low volume flow cell for surface plasmon resonance detection in a miniaturized capillary electrophoresis system

A miniaturized capillary electrophoresis system coupled to a surface plasmon resonance (SPR) sensor on a microfluidic platform fabricated from PDMS is detailed. A previously described split-flow injection technique is first utilized to manipulate sample into the microfluidic chip, followed by separation within the fused-silica capillary and final off-capillary detection of analytes via SPR. Instead of using commercial SPR flow cells requiring relatively large detection volumes, samples of less than 1 nL volume are utilized. The interface between the CE system and SPR sensor made it possible to detect minute volumes of sample with minimal dispersion. The flow cell has the potential to be applicable to miniaturized flow-injection (FI) systems where submicroliter volumes of sample are frequently only available for analysis. The components present in solution, but not bound to the sensor surface, were also investigated. The sensitivity of the CE-SPR system was similar to that found in UV-spectrometric instruments and nonchromophoric components could also be measured.