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1.
Talanta ; 235: 122747, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517615

RESUMO

Microchip capillary electrophoresis (MCE) is a powerful technique for rapid separation; however, its acceptance in routine laboratories is still limited. Compromises caused by the efforts for solving different problems, such as reducing its cost of fabrication and ensuring high separation efficiency, undermine the competitiveness of this technology compared to other separation techniques. Contrary to the conventional pursuit of narrow microchannels, this study investigated the suitability of microchips with channels at the sub-millimeter level, targeting the simplification of the overall operation, cost reduction, and robustness improvement. To this effect, we considered the influence of pressurized flow and Joule heating on the separation. The suppression of pressurized flow with viscous solutions was confirmed through a combination of simulations and experimental results, indicating that the buffer viscosity was enough for successful separation. We fabricated channels of 200 µm × 230 µm using computer numerical controlled (CNC) machining and obtained theoretical plate numbers of 4.8 × 105 m-1 and 5.3 × 105 m-1 for fluorescein isothiocyanate (FITC) labeled small molecules and DNA fragments, respectively, with a buffer viscosity of 168 mPa s (0.5 % hydroxypropyl methylcellulose, HPMC). These values are comparable with that of narrow-bore microchips. Furthermore, we did not observe any deleterious effects with low-conductivity buffers. We investigated the rapid and highly sensitive detection of mycoplasma contamination and the real samples of circulating cell-free DNA (cfDNA), which gave a limit of detection (LOD) as low as 2.3 ng mL-1. Owing to the significant reduction in cost, ease of operation, and fast separation capabilities demonstrated in this work, MCE can be a viable alternative to the usual slab gel electrophoresis running in most biological laboratories.


Assuntos
Eletroforese em Microchip , DNA , Eletroforese Capilar , Derivados da Hipromelose , Limite de Detecção
2.
Clin Chim Acta ; 519: 255-259, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34015305

RESUMO

Cell-free DNA (cfDNA) originates from apoptotic and/or necrotic cells. Few reports are available that examine cfDNA from postmortem samples. Therefore, this study investigated differences between postmortem and biogenic subjects in concentration and fragment distribution of serum cfDNA. We also clarified features of serum cfDNA in postmortem subjects. The results revealed that postmortem subjects had significantly higher cfDNA concentrations than healthy controls and patients with cardiac disease. Serum cfDNA concentrations increased slightly with postmortem interval in subjects who died of asphyxia, and they were slightly higher in subjects who died from internal vs. external causes. Microchip electrophoresis of serum cfDNA revealed a fragment larger than 10,000 bp in only two postmortem subjects; we speculate that the fragment may have originated from necrotic cells. A relatively high concentration of one 150-200 bp fragment was characteristic of postmortem samples. This fragment may have been derived from apoptosis or other processes. We also observed ladder fragments in some subjects who died from external causes. Although additional research is needed for verification, serum cfDNA concentrations and fragment patterns possibly be used as a tool to estimate postmortem intervals and cause of death.


Assuntos
Ácidos Nucleicos Livres , Eletroforese em Microchip , Cardiopatias , Apoptose , Humanos , Necrose
3.
Anal Chem ; 93(13): 5537-5546, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33752328

RESUMO

Sialylated N-glycan isomers with α-2,3 and α-2,6 linkages play crucial and distinctive roles in diverse physiological and pathological processes. Changes of α-2,3-linked sialic acids in sialylated N-glycans are especially important in monitoring the initiation and progression of diseases. However, the specific analysis of α-2,3-sialylated N-glycan linkage isomers remains challenging due to their extremely low abundance and technical limitations in separation and detection. Herein, we designed an integrated strategy that combines linkage-specific derivatization and a charge-sensitive separation method based on microfluidic chip capillary electrophoresis-mass spectrometry (microchip CE-MS) for specific analysis of α-2,3-sialylated N-glycan linkage isomers for the first time. The α-2,6- and α-2,3-sialic acids were selectively labeled with methylamine (MA) and N,N-dimethylethylenediamine (DMEN), respectively, which selectively makes α-2,3-sialylated N-glycans positively charged and realizes online purification, concentration, and discrimination of α-2,3-sialylated N-glycans from other N-glycans in microchip CE-MS. This new approach was demonstrated with standard multisialylated N-glycans, and it was found that only the α-2,3-sialylated N-glycans migrated and were detected in order according to the number of α-2,3-sialic acids. Finally, this strategy was successfully applied in highly sensitive profiling and reproducible quantitation of the serum α-2,3-sialylated N-glycome from ovarian cancer (OC) patients, where 7 of 33 detected α-2,3-sialylated N-glycans significantly changed in the OC group compared with healthy controls.


Assuntos
Eletroforese em Microchip , Polissacarídeos , Eletroforese Capilar , Humanos , Espectrometria de Massas , Ácidos Siálicos
4.
Sensors (Basel) ; 21(4)2021 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-33668587

RESUMO

Staphylococcus aureus (S. aureus) is one of the most common pathogens for nosocomial and community infections, which is closely related to the occurrence of pyogenic and toxic diseases in human beings. In the current study, a lab-built microchip capillary electrophoresis (microchip CE) system was employed for the rapid determination of S. aureus, while a simple-to-use space domain internal standard (SDIS) method was carried out for the reliable quantitative analysis. The precision, accuracy, and reliability of SDIS were investigated in detail. Noted that these properties could be elevated in SDIS compared with traditional IS method. Remarkably, the PCR products of S. aureusnuc gene could be identified and quantitated within 80 s. The theoretical detection limit could achieve a value of 0.066 ng/µL, determined by the using SDIS method. The current work may provide a promising detection strategy for the high-speed and highly efficient analysis of pathogens in the fields of food safety and clinical diagnosis.


Assuntos
Eletroforese em Microchip , Staphylococcus aureus , Eletroforese Capilar , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes
5.
Anal Chem ; 93(7): 3551-3558, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33570925

RESUMO

Current strand displacement amplification (SDA)-based nucleic acid sensing methods generally rely on a ssDNA template that involves complementary bases to the endonuclease recognition sequence, which has the limitation of detecting only short nucleic acids. Herein, a new SDA method in which the defective T junction structure is first used to support SDA (dT-SDA) was proposed and applied in longer DNA detection. In dT-SDA, an auxiliary probe and a primer were designed to specifically identify the target gene, following the formation of a stable defective T junction structure through proximity hybridization, and the formation of defective T junctions could further trigger cascade SDA cycling to produce numerous ssDNA products. The quantity of these ssDNA products was detected through microchip electrophoresis (MCE) and could be transformed to the concentration of the target gene. Moreover, the applicability of this developed strategy in detecting long genomic DNA was verified by detecting bacterial 16S rDNA. This proposed dT-SDA strategy consumes less time and has satisfactory sensitivity, which has great potential for effective bacterial screening and infection diagnosis.


Assuntos
Eletroforese em Microchip , Ácidos Nucleicos , DNA Ribossômico/genética , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico
6.
Anal Bioanal Chem ; 413(11): 3017-3026, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33635387

RESUMO

The long-term consumption of food with pesticide residues has harmful effects on human health and the demand for pesticide detection technology tends to be miniaturized and instant. To this end, we demonstrated the first application of indirectly detecting two carbamate pesticides, metolcarb and carbaryl, by gold nanoparticle-modified indium tin oxide electrode in dual-channel microchip electrophoresis and amperometric detection (ME-AD) system. m-Cresol and α-naphthol were obtained after pesticide hydrolysis in alkaline solution, and then separated and detected by ME-AD. Parameters including the detection potential and running buffer concentration and pH were optimized to improve the detection sensitivity and separation efficiency. Under the optimal conditions, the two analytes were completely separated within 80 s. m-Cresol and α-naphthol presented a wide linear range from 1 to 100 µM, with limits of detection of 0.16 µM and 0.34 µM, respectively (S/N = 3). Moreover, the reliability of this system was demonstrated by analyzing metolcarb and carbaryl in spiked vegetable samples.


Assuntos
Carbamatos/análise , Técnicas Eletroquímicas/métodos , Eletroforese em Microchip/métodos , Resíduos de Praguicidas/análise , Limite de Detecção , Padrões de Referência , Reprodutibilidade dos Testes , Verduras/química
7.
J Chromatogr A ; 1638: 461892, 2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33477027

RESUMO

With advances in the design and fabrication of nanofluidic devices during the last decade, there have been a few reports on nucleic acid analysis using nanoscale electrophoresis. The attractive nature of nanofluidics is the unique phenomena associated with this length scale that are not observed using microchip electrophoresis. Many of these effects are surface-related and include electrostatics, surface roughness, van der Waals interactions, hydrogen bonding, and the electric double layer. The majority of reports related to nanoscale electrophoresis have utilized glass-based devices, which are not suitable for broad dissemination into the separation community because of the sophisticated, time consuming, and high-cost fabrication methods required to produce the relevant devices. In this study, we report the use of thermoplastic nanochannels (110 nm x 110 nm, depth x width) for the free solution electrokinetic analysis of ribonucleotide monophosphates (rNMPs). Thermoplastic devices with micro- and nanofluidic networks were fabricated using nanoimprint lithography (NIL) with the structures enclosed via thermal fusion bonding of a cover plate to the fluidic substrate. Unique to this report is that we fabricated devices in cyclic olefin copolymer (COC) that was thermally fusion bonded to a COC cover plate. Results using COC/COC devices were compared to poly(methyl methacrylate), PMMA, devices with a COC cover plate. Our results indicated that at pH = 7.9, the electrophoresis in free solution resulted in an average resolution of the rNMPs >4 (COC/COC device range = 1.94 - 8.88; PMMA/COC device range = 1.4 - 7.8) with some of the rNMPs showing field-dependent electrophoretic mobilities. Baseline separation of the rNMPs was not possible using PMMA- or COC-based microchip electrophoresis. We also found that COC/COC devices could be assembled and UV/O3 activated after device assembly with the dose of the UV/O3 affecting the magnitude of the electroosmotic flow, EOF. In addition, the bond strength between the substrate and cover plate of unmodified COC/COC devices was higher compared to PMMA/COC devices. The large differences in the electrophoretic mobilities of the rNMPs afforded by nanoscale electrophoresis will enable a new single-molecule sequencing platform we envision, which uses molecular-dependent electrophoretic mobilities to identify the constituent rNMPs generated from an intact RNA molecule using a processive exonuclease. With optimized nanoscale electrophoresis, the rNMPs could be identified via mobility matching at an accuracy >99% in both COC/COC and PMMA/COC devices.


Assuntos
Plásticos/química , Ribonucleotídeos/análise , Eletricidade , Eletro-Osmose , Eletroforese em Microchip , Concentração de Íons de Hidrogênio , Nanotecnologia , Polimetil Metacrilato/química , Água/química
8.
J Chromatogr A ; 1638: 461868, 2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33453653

RESUMO

One of the major drawbacks of electrophoresis in both capillary and microchip is the unsatisfactory sensitivity. Online sample preconcentration techniques can be regarded as the most common and powerful approaches commonly applied to enhance overall detection sensitivity. While the advances of various online preconcentration strategies in capillary and microchip employing aqueous background electrolytes are well-reviewed, there has been limited discussion of the feasible preconcentration techniques specifically developed for capillary and microchip using nonaqueous background electrolytes. This review provides the first consolidated overview of various online preconcentration techniques in nonaqueous capillary and microchip electrophoresis, covering the period of the last two decades. It covers developments in the field of sample stacking, isotachophoresis, and micellar-based stacking. Attention is also given to multi-stacking strategies that have been used for nonaqueous electrophoresis.


Assuntos
Eletroforese Capilar/métodos , Eletroforese em Microchip/métodos , Eletrólitos/química , Isotacoforese , Micelas
9.
Talanta ; 222: 121686, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33167290

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens involved in hospital and community infection. To rapidly and sensitively detect the mecA gene, which is relevant to methicillin-resistant strains, microchip electrophoresis (MCE) integrated with isothermal strand-displacement polymerase reaction (ISDPR) was developed. In the ISDPR signal recycle amplification, the target DNA opened the DNA hairpin structure by specifically binding with the hairpin probe (HP), and then the primer hybridized with the probe and released the target DNA in the presence of Klenow Fragment exo- (KF exo-) polymerase. The released target DNA hybridized with the next HP and then was displaced by the primer again, consequently achieving target recycling and amplification. The amplified products of the HP-cDNA duplex were separated rapidly from other DNAs by MCE. Under optimal conditions, the limit of detection of the target DNA was as low as 12.3 pM (S/N = 3). The proposed ISDPR with MCE method was also successfully applied to detect methicillin-resistant S. aureus, and the experimental results showed that it had some advantages such as being label free, ultrasensitive, rapid and well separated.


Assuntos
Eletroforese em Microchip , Staphylococcus aureus Resistente à Meticilina , Proteínas de Bactérias/genética , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/genética
10.
Talanta ; 224: 121922, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33379123

RESUMO

This review article summarises aspects of the determination of amino acids using capillary and chip electrophoresis in combination with contactless conductivity detection from their historical beginnings to the present time. Discussion is included of the theory of conductivity detection in electromigration techniques, the design of contactless conductivity cells for detection in capillaries and on microchips, including the use of computer programs for simulation of the conductivity response and the process of the electrophoretic separation of amino acids. Emphasis is placed on optimisation of the background electrolyte composition, chiral separation, multidimensional separation, stacking techniques and the use of multidetection systems. There is also a description of clinical applications, the determination of amino acids in foodstuffs, waters, soils and composts with emphasis on modern techniques of sample treatment, such as microdialysis, liquid membrane extraction and many other techniques.


Assuntos
Eletroforese em Microchip , Aminoácidos , Capilares , Condutividade Elétrica , Eletroforese Capilar
11.
Proc Natl Acad Sci U S A ; 117(42): 25985-25990, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33008879

RESUMO

We identify a phenomenon where the onset of channel flow creates an unexpected, charge-dependent accumulation of colloidal particles, which occurs in a common-flow configuration with gas-permeable walls, but in the absence of any installed source of gas. An aqueous suspension of either positively charged (amine-modified polystyrene; a-PS) or negatively charged (polystyrene; PS) particles that flowed into a polydimethylsiloxane (PDMS) channel created charge-dependent accumulation 2 to 4 min after the onset of flow. We unravel the phenomenon with systematic experiments under various conditions and model calculations considering permeability of the channel walls and [Formula: see text]-driven diffusiophoresis. We demonstrate that such spontaneous transport of particles is driven by the gas leakage through permeable walls, which is induced by the pressure difference between the channel and the ambient. Since the liquid pressure is higher, an outward flux of gas forms in the flow. We also observe the phenomenon in a bacterial suspension of Vibrio cholerae, where the fluorescent protein (mKO; monomeric Kusabira Orange) and bacterial cells show charge-dependent separation in a channel flow. Such experimental observations show that diffusiophoresis of charged particles in an aqueous suspension can be achieved by having gas leakage through permeable walls, without any preimposed ion-concentration gradient in the liquid phase. Our findings will help resolve unexpected challenges and biases in on-chip experiments involving particles and gas-permeable walls and help understand similar configurations that naturally exist in physiological systems, such as pulmonary capillaries. We also demonstrate potential applications, such as concentrating and collecting proteins below the isoelectric point.


Assuntos
Dióxido de Carbono/metabolismo , Dimetilpolisiloxanos/química , Proteínas Luminescentes/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Vibrio cholerae/metabolismo , Dióxido de Carbono/análise , Eletroforese em Microchip , Humanos , Técnicas Analíticas Microfluídicas/métodos , Vibrio cholerae/isolamento & purificação
12.
Arch Virol ; 165(12): 2961-2966, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33037940

RESUMO

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). We used microchip electrophoresis in combination with automatic image analysis to develop a novel high-throughput PCR-RFLP to type the gene sequences that encode BLV Tax 233. This method revealed that 233L-Tax is more prevalent than 233P-Tax in cattle in Japan. The proportion infected with BLV carrying the gene encoding 233L-Tax was significantly higher in Holstein cattle than in Japanese Black cattle. Holsteins infected with BLV encoding 233L-Tax had higher proviral loads than did Holsteins infected with BLV encoding 233P-Tax and Japanese Blacks infected with BLV encoding 233L-Tax or 233P-Tax. The novel method developed in this study will be a useful tool for identifying cattle harboring BLV with a higher risk of EBL and viral transmission.


Assuntos
Eletroforese em Microchip/instrumentação , Produtos do Gene tax/genética , Vírus da Leucemia Bovina/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Animais , Bovinos , Leucose Enzoótica Bovina/virologia , Japão , Carga Viral
13.
J Vis Exp ; (159)2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32955503

RESUMO

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that causes progressive loss of muscle mass, leading to premature death. The mutations often cause a distorted reading frame and premature stop codons, resulting in an almost total lack of dystrophin protein. The reading frame can be corrected using antisense oligonucleotides (AONs) that induce exon skipping. The morpholino AON viltolarsen (code name: NS-065/NCNP-01) has been shown to induce exon 53 skipping, restoring the reading frame for patients with exon 52 deletions. We recently administered NS-065/NCNP-01 intravenously to DMD patients in an exploratory investigator-initiated, first-in-human trial of NS-065/NCNP-01. In this methods article, we present the molecular characterization of dystrophin expression using Sanger sequencing, RT-PCR, and western blotting in the clinical trial. The characterization of dystrophin expression was fundamental in the study for showing the efficacy since no functional outcome tests were performed.


Assuntos
Ensaios Clínicos como Assunto , Éxons/genética , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos Antissenso/genética , Anticorpos Bloqueadores/metabolismo , Sequência de Bases , Biópsia , DNA Complementar/genética , Distrofina/genética , Eletroforese em Microchip , Humanos , Músculos/patologia , Mutação/genética , Isoformas de Proteínas/genética , RNA/isolamento & purificação
14.
Electrophoresis ; 41(23): 1961-1968, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32840905

RESUMO

This paper presents an inexpensive and easy-to-implement voltage sequencer instrument for use in microchip capillary electrophoresis (MCE) actuation. The voltage sequencer instrument takes a 0-5 V input signal from a microcontroller and produces a reciprocally proportional voltage signal with the capability to achieve the voltages required for MCE actuation. The unit developed in this work features four independent voltage channels, measures 105 × 143 × 45 mm (width × length × height), and the cost to assemble is under 60 USD. The system is controlled by a peripheral interface controller and commands are given via universal serial bus connection to a personal computer running a command line graphical user interface. The performance of the voltage sequencer is demonstrated by its integration with a fluorescence spectroscopy MCE sensor using pinched sample injection and electrophoretic separation to detect ciprofloxacin in samples of milk. This application is chosen as it is particularly important for the dairy industry, where fines and health concerns are associated with the shipping of antibiotic-contaminated milk. The voltage sequencer instrument presented represents an effective low-cost instrumentation method for conducting MCE, thereby making these experiments accessible and affordable for use in industries such as the dairy industry.


Assuntos
Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Animais , Ciprofloxacina/análise , Resíduos de Drogas/análise , Desenho de Equipamento , Leite/química , Espectrometria de Fluorescência
15.
Anal Chem ; 92(19): 12959-12966, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32842727

RESUMO

There are a variety of complementary observations that could be used in the search for life in extraterrestrial settings. At the molecular scale, patterns in the distribution of organics could provide powerful evidence of a biotic component. In order to observe these molecular biosignatures during spaceflight missions, it is necessary to perform separation science in situ. Microchip electrophoresis (ME) is ideally suited for this task. Although this technique is readily miniaturized and numerous instruments have been developed over the last 3 decades, to date, all lack the automation capabilities needed for future missions of exploration. We have developed a portable, automated, battery-powered, and remotely operated ME instrument coupled to laser-induced fluorescence detection. This system contains all the necessary hardware and software interfaces for end-to-end functionality. Here, we report the first application of the system for amino acid analysis coupled to an extraction unit in order to demonstrate automated sample-to-data operation. The system was remotely operated aboard a rover during a simulated Mars mission in the Atacama Desert, Chile. This is the first demonstration of a fully automated ME analysis of soil samples relevant to planetary exploration. This validation is a critical milestone in the advancement of this technology for future implementation on a spaceflight mission.


Assuntos
Aminoácidos/análise , Automação , Eletroforese em Microchip , Eletroforese em Microchip/instrumentação , Software
16.
Sci Rep ; 10(1): 13548, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782384

RESUMO

Detecting antibiotics in the milk supply chain is crucial to protect humans from allergic reactions, as well as preventing the build-up of antibiotic resistance. The dairy industry has controls in place at processing facilities, but controls on dairy farms are limited to manual devices. Errors in the use of these manual devices can result in severe financial harm to the farms. This illustrates an urgent need for automated methods of detecting antibiotics on a dairy farm, to prevent the shipment of milk containing antibiotics. This work introduces the microchip capillary electrophoresis dairy device, a low-cost system that utilizes microchip capillary electrophoresis as well as fluorescence spectroscopy for the detection of ciprofloxacin contained in milk. The microchip capillary electrophoresis dairy device is operated under antibiotic-absent conditions, with ciprofloxacin not present in a milk sample, and antibiotic-present conditions, with ciprofloxacin present in a milk sample. The response curve for the microchip capillary electrophoresis dairy device is found through experimental operation with varied concentrations of ciprofloxacin. The sensitivity and limit of detection are quantified for the microchip capillary electrophoresis dairy device.


Assuntos
Antibacterianos/análise , Ciprofloxacina/análise , Eletroforese Capilar/métodos , Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Leite/metabolismo , Animais , Antibacterianos/metabolismo , Bovinos , Ciprofloxacina/metabolismo , Leite/química
17.
Anal Methods ; 12(12): 1606-1616, 2020 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661464

RESUMO

Western blotting is a widely used protein assay platform, but the technique requires long analysis times and multiple manual steps. Microfluidic systems are currently being explored for increased automation and reduction of analysis times, sample volumes, and reagent consumption for western blots. Previous work has demonstrated that proteins separated by microchip electrophoresis can be captured on membranes by dragging the microchip outlet across the membrane. This process reduces the separation and transfer time of a western blot to a few minutes. To further improve the speed and miniaturization of a complete western blot, a microscale immunoassay with direct deposition of immunoassay reagents has been developed. Flow deposition of antibodies is used to overcome diffusion limited binding kinetics so that the entire immunoassay can be completed in 1 h with detection sensitivity comparable to incubation steps requiring 20 h. The use of low microliter/min flow rates with antibody reagents applied directly and locally to the membrane where the target proteins have been captured, reduced antibody consumption ~30-fold. The complete western blot was applied to the detection of GAPDH and ß-Tubulin from A431 cell lysate.


Assuntos
Eletroforese em Microchip , Microfluídica , Western Blotting , Imunoensaio , Indicadores e Reagentes
18.
Mikrochim Acta ; 187(8): 448, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32676809

RESUMO

A novel online coupling of microchip isotachophoresis (µITP) with surface-enhanced Raman spectroscopy (SERS) for the analysis of complex samples is presented. Polymeric microchip with coupled channels was used for µITP-SERS analysis of four structurally similar Raman active synthetic dyes (brilliant black BN, carmoisine, ponceau 4R, and sunset yellow FCF) in pharmaceuticals. The µITP separation and simultaneous pre-concentration of the analytes were performed in the first channel of the microchip at pH 6.0 with the aid of non-Raman active discrete spacers (acetate, butyrate, glutarate, pantothenate, and valerate). Silver nanoparticles used for Raman enhancement were present in the second channel, and individual SERS spectra of the dyes were acquired by a mini Raman spectrometer operating at 532 nm. The analytical enhancement factors for silver nanoparticles were 1-5 × 104. The microchip with coupled channels enabled independent µITP separation and SERS detection, and eliminated any adverse impact of nanoparticles on the separation. The developed approach allowed reliable online SERS identification and detection of dyes with limits of detection ranging from 12 to 62 nM. Synthetic dyes were successfully separated, identified, and quantified in pharmaceutical preparations within 7 min without the need for complex or time-consuming sample pretreatment. The results were in good agreement with those obtained by an independent analytical method reported for studied dyes. Graphical abstract.


Assuntos
Corantes/análise , Eletroforese em Microchip/métodos , Isotacoforese/métodos , Análise Espectral Raman/métodos , Limite de Detecção , Nanopartículas Metálicas/química , Prata/química
19.
Electrophoresis ; 41(21-22): 1851-1869, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32530051

RESUMO

Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.


Assuntos
Aldeídos/química , Aminoácidos , Eletroforese Capilar/métodos , Aminoácidos/análise , Aminoácidos/química , Aminoácidos/isolamento & purificação , Eletroforese em Microchip , Naftalenos/química , Estereoisomerismo , o-Ftalaldeído/química
20.
Chem Commun (Camb) ; 56(48): 6579-6582, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32400773

RESUMO

An intracellular signal amplification strategy was developed for the quantification of ATP in single cells by microchip electrophoresis with laser-induced fluorescence detection. By using the method proposed, intracellular ATP levels in single HeLa, HepG2 and HL-7702 cells were found to be in the range of 30-150, 30-140, and 19-120 fmol per cell, respectively.


Assuntos
Trifosfato de Adenosina/análise , Eletroforese em Microchip/métodos , Lasers , Trifosfato de Adenosina/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Linhagem Celular Tumoral , Luciferina de Vaga-Lumes/química , Humanos , Lipossomos/química , Microscopia Confocal , Reprodutibilidade dos Testes , Espectrometria de Fluorescência
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