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.     

电渗流与缓冲液浓度之间数学关系的线性表达

It was believed that electroosmotic mobility μeo is inversely proportional to the square root of the ionic strength L But the linear relationship for regression analysis was expressed differently in different papers. The paper studied the linear expression of the mathematical relationship between μeo and c （background buffer concentration） by mathematical transform and real experimental data.μeo values of fused silica capillary were determined in four buffer systems. Their experimental conditions were controlled carefully for decreasing temperature difference AT and pH difference ApH in 50 μm ID capillary, in which no double layer overlap existed. The linear relationship between the reciprocal of electroosmotic mobility and the square root of concentration （or ionic strength） was derived by mathematical method. The regression analysis of experimental data was shown to well correspond to the relationship. The constants in regression equation could be well defined and the calculated results were acceptable.     

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.     

Effect of alkali metals and metal-ammonia complexes on electroosmosis,effective field strength,and resolution in the separation of diuretics by CZE

The counter ion in CZE separation systems affects resolution, effective field strength and electroosmosis. Alkali metals (lithium, sodium, potassium, and cesium), the ammonium ion, and several complexes of metals with ammonia ([Ag(NH₃)₂]⁺, [Cu(NH₃)₄]²⁺, [Zn(NH₃)₄]²⁺, [Cd(NH₃)₄]²⁺, [Ni(NH₃)₆]²⁺, and [Co(NH₃)₆]²⁺) have been studied for their effect on the separation of diuretics. With the alkali metals the electroosmotic flow velocity decreased and the effective field strength and resolution increased as the hydrated radius of the alkali metal decreased. All the metal-ammonia complexes except that with silver greatly reduced the electroosmotic flow velocity (V_eo) and had only a slight effect on the effective field strength (E_eff). Because these complexes had a negligible effect on the ionic strength of the buffer, they enabled high separating power to be maintained during the separation, and hence the use of more energy in the separation system. This yielded better resolution of the compounds, but the analysis time was then compromised. A simultaneous reduction in capillary length and V_eo while maintaining the high voltage enabled increased resolution without an increase in analysis time. The ability to control V_eo by adding small concentrations (< 100 μM ) of metal complexes to the buffer solution makes it possible to adjust the analysis time and capillary length independently while employing high separation power.     

Fast separation of enantiomers by capillary electrophoresis using a combination of two capillaries with different internal diameters

The combination of capillaries with different internal diameters was used to accelerate the separation of enantiomers in capillary electrophoresis. Separation of R ,S‐1,1′‐binaphthalene‐2,2′‐diyl hydrogen phosphate using isopropyl derivative of cyclofructan 6 was studied as a model system. The best separation conditions included 500 mM sodium borate pH 9.5 with 60 mM concentration of the chiral selector. Separation lasted approx. 1.5 min using the combination of 50 and 100 μm id capillaries of 9.7 cm and 22.9 cm, respectively. It allowed approx. 12‐fold acceleration in comparison to the traditional long‐end separation mainly due to the higher electroosmotic flow generated in the connected capillaries.     

Evaluation of ion mobility in capillary electrophoresis coupled to mass spectrometry for the identification in metabolomics

Currently, feature annotation remains one of the main challenges in untargeted metabolomics. In this context, the information provided by high-resolution mass spectrometry (HRMS) in addition to accurate mass can improve the quality of metabolite annotation, and MS/MS fragmentation patterns are widely used. Accurate mass and a separation index, such as retention time or effective mobility (μ_eff), in chromatographic and electrophoretic approaches, respectively, must be used for unequivocal metabolite identification. The possibility of measuring collision cross-section (CCS) values by using ion mobility (IM) is becoming increasingly popular in metabolomic studies thanks to the new generation of IM mass spectrometers. Based on their similar separation mechanisms involving electric field and the size of the compounds, the complementarity of ^DTCCS_N2 and μ_eff needs to be evaluated. In this study, a comparison of ^DTCCS_N2 and μ_eff was achieved in the context of feature identification ability in untargeted metabolomics by capillary zone electrophoresis (CZE) coupled with HRMS. This study confirms the high correlation of ^DTCCS_N2 with the mass of the studied metabolites as well as the orthogonality between accurate mass and μ_eff, making this combination particularly interesting for the identification of several endogenous metabolites. The use of IM-MS remains of great interest for facilitating the annotation of neutral metabolites present in the electroosmotic flow (EOF) that are poorly or not separated by CZE.     

Rapid and cost‐effective method for the simultaneous quantification of seven alkaloids in Corydalis decumbens by microwave‐assisted extraction and capillary electrophoresis

A rapid and cost‐effective method based on microwave‐assisted extraction followed by capillary electrophoresis was developed for simultaneous quantification of seven alkaloids in Corydalis decumbens for the first time. The main parameters affecting microwave‐assisted extraction and capillary electrophoresis separation were investigated and optimized. The optimal microwave‐assisted extraction was performed at 40°C for 5 min using methanol/water (90:10, v/v) as the extracting solvent. Electrophoretic separation was achieved within 15 min using an uncoated fused‐silica capillary (50 μm internal diameter and 27.7 cm effective length) and a 500 mM Tris buffer containing 45% v/v methanol (titrated to pH^* 2.86 with H₃PO₄). The developed method was successfully applied to the quantification of seven alkaloids in Corydalis decumbens obtained from different regions of China. The combination of microwave‐assisted extraction with capillary electrophoresis was an effective method for the rapid analysis of the alkaloids in Corydalis decumbens .     

Characterization of a capacitance-coupled contactless conductivity detection system with sidewall electrodes on a low-voltage-driven electrophoresis microchip

A new type of capacitance-coupled contactless conductivity detection (C⁴D) system with sidewall electrodes was proposed for integration on a silicon-on-isolator–poly(dimethylsiloxane) (SOI-PDMS) hybrid low-voltage-driven electrophoresis microchip. By a microelectromechanical system process, the sidewall electrodes were fabricated precisely at either side of the separation channel. The area of the capacitor electrodes was the maximum value to improve the detection sensitivity with an enhanced capacitance effect. According to the simulation results, the structural parameters of the sidewall electrodes were determined as 550-μm length, 15-μm width, 80-μm separation distance, and 1-μm isolator thickness. The integrated microdevice with the SOI-PDMS hybrid electrophoresis microchip was very compact and the size was only 15 cm × 15 cm × 10 cm (width × length × height), which permitted miniaturization and portability. The detector performance was evaluated by K⁺ testing. The detection limit of the conductivity detector was determined to be 10^-9 and 10^-6 M for K⁺ in the static and electric-driven modes, respectively. Finally, the C⁴D was applied to low-voltage-driven electrophoresis on a microchip to carry out real-time measurement of the separation of amino acids. The separations of 10^-4 M lysine and phenylalanine in the low-voltage-driven electrophoresis mode were performed with an electric field of 300 V/cm and were completed in less than 15 min with a resolution of 1.3. The separation efficiency was found to be 1.3 × 10³ and 2.8 × 10³ plates for lysine and phenylalanine, respectively, with a migration time reproducibility of 2.7 and 3.2%. The conductivity detection limit of amino acids achieved was 10^-6 M. The proposed method for the construction of a novel C⁴D integrated on an SOI-PDMS hybrid low-voltage-driven electrophoresis microchip showed the most extensive integration and miniaturization of a microdevice, which is a further crucial step toward the realization of the “lab-on-a-chip” concept.     

Capillary electrophoresis microchip coupled with on-line chemiluminescence detection

In the present work, chemiluminescence detection was integrated with capillary electrophoresis microchip. The microchip was designed on the principle of flow-injection chemiluminescence system and capillary electrophoresis. It has three main channels, five reservoirs and a detection cell. As model samples, dopamine and catechol were separated and detected using a permanganate chemiluminescent system on the prepared microchip. The samples were electrokinetically injected into the double-T cross section, separated in the separation channel, and then oxidized by chemiluminescent reagent delivered by a home-made micropump to produce light in the detection cell. The electroosmotic flow could be smoothly coupled with the micropump flow. The detection limits for dopamine and catechol were 20.0 and 10.0 μM, respectively. Successful separation and detection of dopamine and catechol demonstrated the distinct advantages of integration of chemiluminescent detection on a microchip for rapid and sensitive analysis.     

Rapid Determination of Creatinine in Human Urine by Microchip Electrophoresis with LED Induced Fluorescence Detection

In clinical medicine, urine creatinine concentration is an important marker in the evaluation of renal function and muscular dysfunctions. Herein, we reported a novel method for rapid determination of creatinine in urine by microchip electrophoresis with light-emitting diode induced fluorescence detection. Creatinine was derivatized by fluorescein isothiocyanate, and then quantitatively detected by the developed microchip LED induced fluorescence detection system. The excitation and emission wavelengths were 490 and 523 nm, respectively. The urine samples were analyzed after centrifuge and filtration. A baseline separation was obtained in <30 s using 10 mM borate buffer (pH 9.0, containing 45 mM sodium dodecylsulfate), with separation voltage of 1.5 kV. Good linearity was obtained (r ² = 0.9978) in the concentration range of 10.0–2.00 × 10³ μM, and the limit of detection was 2.87 μM (S/N = 3). The recovery was 96.0–107 %, and the interday precision was <4.5 % (n = 6). To validate assay results, we compared the present method with the Jaffe’s colorimetric assay by measuring real urine samples. The method was reliable, sensitive, high-speed, low-cost and suitable for the routine analysis of creatinine in biofluids.

     

Integration of a flow-type chemiluminescence detector on a glass electrophoresis chip

A glass electrophoresis microchip integrated a flow-type chemiluminescence (CL) detection cell has been developed and evaluated. The chip pattern is a double-T-type electrophoretic sample injection and separation combining with a Y-type chemiluminecent detector. The double-T geometry allows for high-efficiency sample injection and geometric definition of sample plug size. The branch of Y was used as CL reagent channel, and the CL reagent was delivered by a lab-made micropump. Bis[(2,4,6-trichlorophenyl)]oxalate-H₂O₂ CL system was employed to detect dansyl amino acids. On this microchip, dansyl-phenylalanine and -sarcosine were successfully separated by electrophoresis and detected within 250 s. The detection limits (S/N=3) of dansyl-phenylalanine and -sarcosine could reach to 2.8 and 3.2 μM, respectively, due to the vigorous dilution of sample with CL reagent and timely removal of the waste solution from reaction area.     

Integrated microfluidic device for the separation and electrochemical detection of catechol estrogen-derived DNA adducts

Catechol estrogen-derived DNA adducts are formed as a result of the reaction of catechol estrogen metabolites (e.g., catechol estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various catechol estrogen-derived DNA adducts. A hybrid PDMS/glass microchip with in-channel amperometric detection interfaced with in situ palladium decoupler is utilized and presented. The influence of buffer additives along with the effect of the separation voltage on the resolving power of the microchip is discussed. Calibration plots were constructed in the range 0.4–10 μM with r ² ≥ 0.999, and detection limits in the attomole range are reported. These results suggest that on-chip analysis is applicable for analyzing various DNA adducts as potential biomarkers for future medical diagnostics.     

Rapid determination of NSAIDs by capillary and microchip electrophoresis with capacitively coupled contactless conductivity detection in wastewater

A simple, rapid method using CE and microchip electrophoresis with C⁴D has been developed for the separation of four nonsteroidal anti-inflammatory drugs (NSAIDs) in the environmental sample. The investigated compounds were ibuprofen (IB), ketoprofen (KET), acetylsalicylic acid (ASA), and diclofenac sodium (DIC). In the present study, we applied for the first time microchip electrophoresis with C⁴D detection to the separation and detection of ASA, IB, DIC, and KET in the wastewater matrix. Under optimum conditions, the four NSAIDs compounds could be well separated in less than 1 min in a BGE composed of 20 mM His/15 mM Tris, pH 8.6, 2 mM hydroxypropyl-beta-cyclodextrin, and 10% methanol (v/v) at a separation voltage of 1000–1200 V. The proposed method showed excellent repeatability, good sensitivity (LODs ranging between 0.156 and 0.6 mg/L), low cost, high sample throughputs, portable instrumentation for mobile deployment, and extremely lower reagent and sample consumption. The developed method was applied to the analysis of pharmaceuticals in wastewater samples with satisfactory recoveries ranging from 62.5% to 118%.     

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.     

Determination of rhein and physcion in rhubarb by microchip capillary electrophoresis in mixed hydro-organic solvent combined with laser-induced fluorescence detection

Microchip capillary electrophoresis in mixed hydro-organic solvent combined with laser-induced fluorescence detection was developed for the separation and detection of physcion and rhein in rhubarb. In contrast to the conventional capillary electrophoresis method, ammonium acetate-dimethyl sulfoxide was used as the basic buffer system in this method. The effects of background buffer, buffer apparent pH*, buffer concentration, water ratio, sample preparation method, and separation voltage on separation and detection were investigated. Optimized separation and detection conditions were obtained: the buffer consisted of 20 mmol/L of ammonium acetate in hydro-organic solvent composed dimethyl sulfoxide, formamide, and water mixed at 60/20/20 (v/v/v) ratio. The separation voltage was 1.9 kV. Under these conditions, the physcion, rhein, and other components of rhubarb can be completely separated within 150 s. Under the methodological verification, good linearity (R ≥ 0.9995) for physcion and rhein, and low limits of detection (0.085 μg·mL⁻¹ and 0.077 μg·mL⁻¹, respectively), satisfactory peak area precisions, migration time precisions (1.74%–3.09%), and accuracy (recovery rate 97.8% and 101.4%) were achieved. It is shown that the proposed method is simple, efficient, fast, sensitive, simple instrument, consumes few samples, has low operating cost, and is linear.     

Fast analysis of domoic acid using microchip electrophoresis with laser‐induced fluorescence detection

A fast and effective method was developed to detect domoic acid based upon microchip electrophoresis combined with laser‐induced fluorescence detection. Through study of the gated injection process on the cross channel of the microchip, the low‐voltage mode with relatively longer sample loading time was adopted to reduce the sample discrimination and improve the signal sensitivity. Fluorescein isothiocyanate was used as the derivatizing reagent for domoic acid. Under the optimized conditions, domoic acid was completely separated in 60 s with separation efficiency of 1.35 × 10⁵ m⁻¹. The calibration curve was obtained in the range of 1.0 × 10⁻⁹ to 1.0 × 10⁻⁷ mol/L, and the detection limit reached 2.8 × 10⁻¹⁰ mol/L. This developed method was successfully applied to analyze domoic acid in real samples.     

Reduction in sample injection bias using pressure gradients generated on chip

Sample injection in microchip-based capillary zone electrophoresis (CZE) frequently rely on the use of electric fields which can introduce differences in the injected volume for the various analytes depending on their electrophoretic mobilities and molecular diffusivities. While such injection biases may be minimized by employing hydrodynamic flows during the injection process, this approach typically requires excellent dynamic control over the pressure gradients applied within a microfluidic network. The current article describes a microchip device that offers this needed control by generating pressure gradients on-chip via electrokinetic means to minimize the dead volume in the system. In order to realize the desired pressure-generation capability, an electric field was applied across two channel segments of different depths to produce a mismatch in the electroosmotic flow rate at their junction. The resulting pressure-driven flow was then utilized to introduce sample zones into a CZE channel with minimal injection bias. The reported injection strategy allowed the introduction of narrow sample plugs with spatial standard deviations down to about 45 μm. This injection technique was later integrated to a capillary zone electrophoresis process for analyzing amino acid samples yielding separation resolutions of about 4–6 for the analyte peaks in a 3 cm long analysis channel.     

Covalent anionic copolymer coatings with tunable electroosmotic flow for optimization of capillary electrophoretic separations

We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40 × 10⁻⁹ m² V⁻¹ s⁻¹ interval for PAMAMPS coatings ranging from 0 to 60 mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2–10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.     

Rapid separation of post‐blast explosive residues on glass electrophoresis microchips

This study describes the development of an analytical methodology based on the use of microchip electrophoresis (ME) devices integrated with capacitively coupled contactless conductivity detection (C⁴D) for the separation and detection of inorganic anions in post‐blast explosive residues. The best separation condition was achieved using a running buffer composed of 35 mmol/L lactic acid, 10 mmol/L histidine and 0.070 mmol/L cetyl(trimethyl ammonium) bromide. For C⁴D measurements, the highest sensitivity was obtained applying a 700 kHz sinusoidal wave with excitation voltage of 20 V_pp. The separation of Cl^?, NO₃^?, NO₂^?, SO₄^2?, ClO₄^? and ClO₃^? was performed within ca. 150 s with baseline resolution and efficiencies between 4.4 × 10⁴ and 1.7 × 10⁵ plates/m. The found limits of detection ranged between 2.5 and 9.5 μmol/L. Last, real samples of post‐blast explosive residues were analyzed on the ME‐C⁴D devices obtaining successfully the determination of Cl^?, NO₃^? and SO₄^2?. The achieved concentration values varied between 12.8–72.5 mg/L for Cl^?, 1.7–293.1 mg/L for NO₃^? and 1.3–201.3 mg/L for SO₄^2?. The data obtained using ME‐C⁴D devices were in good agreement with the concentrations found by ion chromatography. The approach reported herein has provided short analysis time, instrumental simplicity, good analytical performance and low cost. Furthermore, the ME‐C⁴D devices emerge as a powerful and portable analytical platform for on‐site analysis demonstrating to be a promising tool for the crime scene investigation.     

Fast quantification of amino acids by microchip electrophoresis–mass spectrometry

A fast microchip electrophoresis–nano-electrospray ionization-mass spectrometric method (MCE-nanoESI-MS) was developed for analysis of amino acids in biological samples. A glass/poly(dimethylsiloxane) hybrid microchip with a monolithic nanoESI emitter was used in the platform. The proposed MCE-nanoESI-MS analytical method showed high separation efficiency for amino acids. Baseline separation of an amino acid mixture containing Lys, Arg, Val, Tyr, and Glu was completed within 120 s with theoretical plate numbers of >7,500. The method was applied to study cellular release of excitatory amino acids (i.e., aspartic acid (Asp) and glutamic acid (Glu)) under chemical stimulations. Linear calibration curves were obtained for both Asp and Glu in a concentration range from 1.00 to 150.0 μM. Limits of detection were found to be 0.37 μM for Asp and 0.33 μM for Glu (S/N?=?3). Assay repeatability (relative standard deviation, n?=?6) was 4.2 and 4.5 %, for Asp and Glu at 5.0 μM, respectively. In the study of cellular release, PC-12 nerve cells were incubated with alcohol at various concentrations for 1 h. Both extra- and intracellular levels of Asp and Glu were measured by the proposed method. The results clearly indicated that ethanol promoted the release of both Asp and Glu from the cells.