RESUMEN
Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.
Asunto(s)
Senescencia Celular/fisiología , Resistencia a Medicamentos/fisiología , Fenotipo , Factor de Transcripción ReIA/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/fisiología , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma/metabolismo , Ratones , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Interferente Pequeño/metabolismo , Tetraciclina/farmacología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
A critical challenge to translating field effect transistors into biochemical sensor platforms is the requirement of a gate electrode, which imposes restrictions on sensor device architectures and results in added expense, poorer scalability, and electrical noise. Here we show that it is possible to eliminate the need of the physical gate electrode and dielectrics altogether using a synthetic tube-in-a-tube (Tubeâ§2) semiconductor. Composed of a semiconducting single-walled carbon nanotube nested in a charged, impermeable covalent functional shell, Tubeâ§2 allows the semiconducting conduction pathway to be modulated solely by surface functional groups in a chemically gated-all-around configuration. The removal of physical gates significantly simplifies the device architecture and enables photolithography-free, highly scalable fabrication of transistor sensors in nonconventional configurations that are otherwise impossible. We show that concomitant FET sensitivity and single-mismatch selectivity can be achieved with Tubeâ§2 even in a two-terminal, thin film transistor device configuration that is as simple as a chemiresistor. Miniaturized two-terminal field effect point sensors can also be fabricated, using a straightforward dice-and-dip procedure, for the detection of tuberculosis biomarkers.
Asunto(s)
Nanotubos de Carbono/química , Biomarcadores/análisis , Compuestos de Diazonio/química , Conductividad Eléctrica , Electrodos , Humanos , Técnicas Analíticas Microfluídicas , Oligonucleótidos/análisis , Semiconductores , Tuberculosis/diagnósticoRESUMEN
Single-walled carbon nanotubes (SWCNTs) hold vast potential for future electronic devices due to their outstanding properties, however covalent functionalization often destroys the intrinsic properties of SWCNTs, thus limiting their full potential. Here, we demonstrate the fabrication of a functionalized graphene/semiconducting SWCNT (T@fG) heterostructured thin film transistor as a chemical sensor. In this structural configuration, graphene acts as an atom-thick, impermeable layer that can be covalently functionalized via facile diazonium chemistry to afford a high density of surface functional groups while protecting the underlying SWCNT network from chemical modification, even during a covalent chemical reaction. As a result, the highly functionalized carbon-based hybrid structure exhibits excellent transistor properties with a carrier mobility and ON/OFF ratio as high as 64 cm2/Vs and 5400, respectively. To demonstrate its use in potential applications, T@fG thin films were fabricated as aqueous ammonium sensors exhibiting a detection limit of 0.25 µM in a millimolar ionic strength solution, which is comparable with state-of-the-art aqueous ammonium nanosensors.
RESUMEN
Covalently functionalized, semiconducting double-walled carbon nanotubes exhibit remarkable properties and can outperform their single-walled carbon nanotube counterparts. In order to harness their potential for electronic applications, metallic double-walled carbon nanotubes must be separated from the semiconductors. However, the inner wall is inaccessible to current separation techniques which rely on the surface properties. Here, the first approach to address this challenge through electrical breakdown of metallic double-walled carbon nanotubes, both inner and outer walls, within networks of mixed electronic types is described. The intact semiconductors demonstrate a â¼62% retention of the ON-state conductance in thin film transistors in response to covalent functionalization. The selective elimination of the metallic pathways improves the ON/OFF ratio, by more than 360 times, to as high as 40 700, while simultaneously retaining high ON-state conductance.
Asunto(s)
Metales/química , Nanotubos de Carbono/química , Benceno/química , Electricidad , Factores de Tiempo , Transistores ElectrónicosRESUMEN
A hybrid microchip/capillary electrophoresis (CE) system was developed to allow unbiased and lossless sample loading and high-throughput repeated injections. This new hybrid CE system consists of a poly(dimethylsiloxane) (PDMS) microchip sample injector featuring a pneumatic microvalve that separates a sample introduction channel from a short sample loading channel, and a fused-silica capillary separation column that connects seamlessly to the sample loading channel. The sample introduction channel is pressurized such that when the pneumatic microvalve opens briefly, a variable-volume sample plug is introduced into the loading channel. A high voltage for CE separation is continuously applied across the loading channel and the fused-silica capillary separation column. Analytes are rapidly separated in the fused-silica capillary, and following separation, high-sensitivity MS detection is accomplished via a sheathless CE/ESI-MS interface. The performance evaluation of the complete CE/ESI-MS platform demonstrated that reproducible sample injection with well controlled sample plug volumes could be achieved by using the PDMS microchip injector. The absence of band broadening from microchip to capillary indicated a minimum dead volume at the junction. The capabilities of the new CE/ESI-MS platform in performing high-throughput and quantitative sample analyses were demonstrated by the repeated sample injection without interrupting an ongoing separation and a linear dependence of the total analyte ion abundance on the sample plug volume using a mixture of peptide standards. The separation efficiency of the new platform was also evaluated systematically at different sample injection times, flow rates, and CE separation voltages.
Asunto(s)
Electroforesis por Microchip/instrumentación , Análisis de Inyección de Flujo/instrumentación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Dimetilpolisiloxanos/química , Diseño de Equipo , Hidrodinámica , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
Atom-thick materials such as single-walled carbon nanotubes (SWCNTs) and graphene exhibit ultrahigh sensitivity to chemical perturbation partly because all of the constituent atoms are surface atoms. However, low selectivity due to nonspecific binding on the graphitic surface is a challenging issue to many applications including chemical sensing. Here, we demonstrated simultaneous attainment of high sensitivity and selectivity in thin-film field effect transistors (TFTs) based on outer-wall selectively functionalized double-walled carbon nanotubes (DWCNTs). With carboxylic acid functionalized DWCNT TFTs, we obtained excellent gate modulation (on/off ratio as high as 4000) with relatively high ON currents at a CNT areal density as low as 35 ng/cm(2). The devices displayed an NH(3) sensitivity of 60 nM (or ~1 ppb), which is comparable to small molecule aqueous solution detection using state-of-the-art SWCNT TFT sensors while concomitantly achieving 6000 times higher chemical selectivity toward a variety of amine-containing analyte molecules over that of other small molecules. These results highlight the potential of using covalently functionalized double-walled carbon nanotubes for simultaneous ultrahigh selective and sensitive detection of chemicals and illustrate some of the structural advantages of this double-wall materials strategy to nanoelectronics.
Asunto(s)
Amoníaco/análisis , Electrónica , Nanotubos de Carbono/química , Ácidos Carboxílicos/química , Grafito/química , Propiedades de SuperficieRESUMEN
A novel sheathless capillary isotachophoresis (CITP/CZE)-mass spectrometry (MS) interface featuring a large inner diameter (i.d.) separation capillary, and a detachable small i.d. porous electrospray ionization (ESI) emitter was developed in this study to simultaneously achieve large sample loading capacity and stable nanoESI operation. Crucial operating parameters, including sample loading volume, flow rate, and separation window, were systematically investigated to attain optimum CITP/CZE separation efficiency and MS detection sensitivity. The performance of CITP/CZE-nanoESI-MS using the new sheathless interface was evaluated for its achievable low limit of quantification (LOQ) by analyzing targeted peptides, leu-enkephalin and angiotensin II, spiked in a BSA tryptic digest matrix at different concentrations. A linear dynamic range spanning 4.5 orders of magnitude and a 10 pM LOQ with measurement reproducibility of the CV < 22% were obtained experimentally for both targeted peptides, representing a 5-fold sensitivity improvement as compared to using the sheath liquid interface developed previously.1.
Asunto(s)
Isotacoforesis/métodos , Nanotecnología/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Péptidos/análisis , Péptidos/químicaRESUMEN
Complicating proteomic analysis of whole tissues is the obvious problem of cell heterogeneity in tissues, which often results in misleading or confusing molecular findings. Thus, the coupling of tissue microdissection for tumor cell enrichment with capillary isotachophoresis-based selective analyte concentration not only serves as a synergistic strategy to characterize low abundance proteins, but it can also be employed to conduct comparative proteomic studies of human astrocytomas. A set of fresh frozen brain biopsies were selectively microdissected to provide an enriched, high quality, and reproducible sample of tumor cells. Despite sharing many common proteins, there are significant differences in the protein expression level among different grades of astrocytomas. A large number of proteins, such as plasma membrane proteins EGFR and Erbb2, are up-regulated in glioblastoma. Besides facilitating the prioritization of follow-on biomarker selection and validation, comparative proteomics involving measurements in changes of pathways are expected to reveal the molecular relationships among different pathological grades of gliomas and potential molecular mechanisms that drive gliomagenesis.
Asunto(s)
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteoma/metabolismo , Astrocitoma/patología , Neoplasias Encefálicas/patología , Cromatografía de Fase Inversa , Análisis por Conglomerados , Electroforesis Capilar , Redes Reguladoras de Genes , Humanos , Inmunohistoquímica , Captura por Microdisección con Láser , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Estadificación de Neoplasias , Mapas de Interacción de Proteínas , Proteoma/genética , Proteoma/aislamiento & purificación , Proteómica , Análisis de Matrices Tisulares , Regulación hacia ArribaRESUMEN
We demonstrate the direct coupling of transient capillary isotachophoresis/capillary zone electrophoresis (CITP/CZE) with a high-sensitivity triple quadrupole mass spectrometer operating in selected reaction monitoring (SRM) mode for sample quantitation. The capability of CITP/CZE for in situ sample enrichment and separation has been shown to significantly improve the analytical figures of merit. A linear dynamic range spanning 4 orders of magnitude was observed. An average signal-to-noise ratio (S/N) of 49.6 was observed for 50 amol of targeted peptide in the presence of a complex and much more abundant bovine serum albumin (BSA) digest. Correlation of variation (CV) of <10% for peak area was measured from triplicate sample analyses at 50 pM peptide concentration, showing good reproducibility of this online CITP/CZE-SRM mass spectrometry (MS) platform, and with limit of quantitation (LOQ) demonstrated to be well below 50 pM.
Asunto(s)
Electroforesis Capilar , Isotacoforesis , Fragmentos de Péptidos/análisis , Albúmina Sérica Bovina/análisis , Espectrometría de Masa por Ionización de Electrospray , Animales , Bovinos , Humanos , Reproducibilidad de los Resultados , Relación Señal-RuidoRESUMEN
A two-dimensional microfluidic system is presented for intact protein separations combining isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) employing in situ photopolymerized polyacrylamide (PAAm) gels. The PAAm gels are used for multiple functions. In addition to serving as a highly-resolving separation medium for gel electrophoresis, discrete polyacrylamide gel plugs are used to enable the efficient isolation of different on-chip media including anolyte, catholyte, and sample/ampholyte solutions for IEF. The gel plugs are demonstrated as on-chip reagent containers, holding defined quantities of SDS for on-chip SDS-protein complexation, and enabling the use of a discontinuous buffer system for sample band sharpening during SDS-PAGE. The 2-D chip also employs several unique design features including an angled isoelectric focusing channel to minimize sample tailing, and backbiasing channels designed to achieve uniform interdimensional sample transfer. Separation results using E. coli cell lysate are presented using a 10-channel chip with and without the discontinuous buffer system, with resolving power more than doubled in the former case. Further improvements in separation resolution are demonstrated using a 20-channel chip design.
Asunto(s)
Electroforesis por Microchip/instrumentación , Electroforesis en Gel de Poliacrilamida/instrumentación , Focalización Isoeléctrica/instrumentación , Proteínas/análisis , Resinas Acrílicas/química , Tampones (Química) , Electroforesis en Gel Bidimensional/instrumentación , Diseño de Equipo , Escherichia coli , Reproducibilidad de los ResultadosRESUMEN
A compelling need exists for the development of technologies that facilitate and accelerate the discovery of novel protein biomarkers with therapeutic and diagnostic potential. The inherent disadvantage of biomarker dilution in complex biological fluids such as serum/plasma, urine, and saliva necessitates highly sensitive analytical approaches, often exceeding the dynamic range of currently available proteomic platforms. Thus, investigative studies directed at tissues obtained from the primary site of pathology probably afford the best opportunity for the discovery of disease biomarkers. This review therefore focuses on the most recent advances in capillary electrophoresis-based single and multidimensional separations coupled with ESI-MS for performing comprehensive and comparative analysis of protein expression profiles within clinical specimens. Advanced sample preparation techniques, including tissue microdissection, detergent-based membrane protein extraction, and heat-induced protein retrieval, further enable targeted protein profiling of both fresh-frozen, formalin-fixed, and paraffin-embedded tissues. Comparative proteomics involving measurements in changes of biological pathways or functional processes are expected to provide relevant disease-associated markers and networks, molecular relationships among different stages of disease, and molecular mechanisms that drive the progression of disease. From a practical perspective, the evaluation of comparative proteomic dataset within a biological context is essential for high-throughput data validation, prioritization of follow-on biomarker selection, and validation experiments.
Asunto(s)
Biomarcadores/análisis , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Proteómica/métodos , Animales , HumanosRESUMEN
A compelling need exists for the development of technologies that facilitate and accelerate the discovery of novel protein biomarkers with therapeutic and diagnostic potential. Comparisons among shotgun proteome technologies, including capillary isotachophoresis (CITP)-based multidimensional separations and multidimensional LC system, are therefore performed in this study regarding their abilities to address the challenges of protein complexity and relative abundance inherent in glioblastoma multiforme-derived cancer stem cells. Comparisons are conducted using a single processed protein digest with equal sample loading, identical second-dimension separation (RPLC) and MS conditions, and consistent search parameters and cutoff established by the target-decoy determined false-discovery rate. Besides achieving superior overall proteome performance in total peptide, distinct peptide, and distinct protein identifications; analytical reproducibility of the CITP proteome platform coupled with the spectral counting approach are determined by a Pearson R(2) value of 0.98 and a CV of 15% across all proteins quantified. In contrast, extensive fraction overlapping in strong cation exchange greatly limits the ability of multidimensional LC separations for mining deeper into the tissue proteome as evidenced by the poor coverage in various protein functional categories and key protein pathways. The CITP proteomic technology, equipped with selective analyte enrichment and ultrahigh resolving power, is expected to serve as a critical component in the overall toolset required for biomarker discovery via shotgun proteomic analysis of tissue specimens.
Asunto(s)
Biomarcadores/análisis , Mapeo Peptídico/métodos , Proteómica/métodos , Cromatografía por Intercambio Iónico , Electroforesis Capilar , Quinasas MAP Reguladas por Señal Extracelular/análisis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glioblastoma , Humanos , Focalización Isoeléctrica , Sistema de Señalización de MAP Quinasas , Espectrometría de Masas , Células Madre Neoplásicas/química , Células Madre Neoplásicas/enzimología , Neuronas/química , Neuronas/metabolismo , Especificidad de Órganos , Reproducibilidad de los Resultados , Células Tumorales CultivadasRESUMEN
The vast number of proteins present in the proteome of a typical organism requires that separations be performed on the mixture prior to introduction into a mass spectrometer for protein identification and quantification. An integrated protein separation platform, combining capillary isoelectric focusing (CIEF) with reversed phase liquid chromatography (RPLC), is described to provide high resolving power for the analysis of complex protein mixtures. Thus, the proteins are systematically resolved according to their differences in isoelectric point and hydrophobicity using combined CIEF/RPLC separations. A key feature of the CIEF-based multidimensional separation platform is the elimination of protein loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein profiles within small cell populations or limited tissue samples.
Asunto(s)
Cromatografía Liquida/métodos , Focalización Isoeléctrica/métodos , Espectrometría de Masas/métodos , Métodos Analíticos de la Preparación de la Muestra , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , SolubilidadRESUMEN
Dynamic changes in the expression of multiple genes appear to be common features that distinguish transformed cells from their normal counterparts. We compared the proteomic profiles of four glioblastoma multiforme (GBM) tissue samples and four normal brain cortex samples to examine the molecular basis of gliomagenesis. Trypsin-digested protein samples were separated by capillary isoelectric focusing with nano-reversed-phase liquid chromatography and were profiled by mass spectrometric sequencing. Wolf-Hirschhorn syndrome candidate 1 (WHSC1), along with 103 other proteins, was found only in the GBM proteomes. Western blot and immunohistochemistry verified our proteomic findings and demonstrated that 30-kDa WHSC1 expression increases with ascending tumor proliferation activity. RNA interference could suppress glioma cell growth by blocking WHSC1 expression. Our novel findings encourage the application of proteomic techniques in cancer research.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glioma/metabolismo , Glioma/patología , N-Metiltransferasa de Histona-Lisina/biosíntesis , Proteínas Represoras/biosíntesis , Western Blotting , Proliferación Celular , Cromatografía Liquida , Humanos , Inmunohistoquímica , Proteómica , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , TransfecciónRESUMEN
Targeted proteomics research, based on the enrichment of disease-relevant proteins from isolated cell populations selected from high-quality tissue specimens, offers great potential for the identification of diagnostic, prognostic, and predictive biological markers for use in the clinical setting and during preclinical testing and clinical trials, as well as for the discovery and validation of new protein drug targets. Formalin-fixed and paraffin-embedded (FFPE) tissue collections, with attached clinical and outcome information, are invaluable resources for conducting retrospective protein biomarker investigations and performing translational studies of cancer and other diseases. Combined capillary isoelectric focusing/nano-reversed-phase liquid chromatography separations equipped with nano-electrospray ionization-tandem mass spectrometry are employed for the studies of proteins extracted from microdissected FFPE glioblastoma tissues using a heat-induced antigen retrieval (AR) technique. A total of 14,478 distinct peptides are identified, leading to the identification of 2733 non-redundant SwissProt protein entries. Eighty-three percent of identified FFPE tissue proteins overlap with those obtained from the pellet fraction of fresh-frozen tissue of the same patient. This large degree of protein overlapping is attributed to the application of detergent-based protein extraction in both the cell pellet preparation protocol and the AR technique.
Asunto(s)
Proteoma/análisis , Secuencia de Aminoácidos , Neoplasias Encefálicas/metabolismo , Cromatografía Liquida , Reacciones Falso Positivas , Fijadores , Formaldehído , Glioblastoma/metabolismo , Humanos , Focalización Isoeléctrica , Microdisección , Datos de Secuencia Molecular , Adhesión en Parafina , Péptidos/análisis , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.
Asunto(s)
Análisis Mutacional de ADN/métodos , Electroforesis en Gel Bidimensional/métodos , Pruebas Genéticas , Microfluídica/métodos , Polímeros/química , ADN/química , Análisis Mutacional de ADN/instrumentación , Electroforesis en Gel Bidimensional/instrumentación , Análisis de Elementos Finitos , Concentración de Iones de Hidrógeno , Microfluídica/instrumentación , Modelos Teóricos , Mutación Puntual , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Temperatura , Factores de TiempoRESUMEN
The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.
Asunto(s)
Electroforesis Capilar/métodos , Focalización Isoeléctrica/métodos , Proteómica/métodos , Humanos , Péptidos/química , Péptidos/aislamiento & purificaciónRESUMEN
For top-down proteomics, nano-reversed phase liquid chromatography (RPLC) plays a major role in both single and multidimensional protein separations in an effort to increase the overall peak capacity for the resolution of complex protein mixtures prior to mass spectrometry analysis. Effects of various chromatography conditions, including alkyl chain length in the stationary phase, capillary column temperature, and ion-pairing agent, on the resolution of intact proteins are studied using nano-RPLC-electrospray ionization-mass spectrometry. Optimal chromatography conditions include the use of C18 column heated at 60 degrees C and the addition of trifluoroacetic acid instead of heptafluorobutyric acid as the ion-paring agent in the mobile phase. Under optimized chromatography conditions, there are no significant differences in the separation performance of yeast cell lysates present in the native versus denatured states. Denatured yeast proteins resolved and eluted from nano-RPLC can be subjected to proteolytic digestion in an on- or off-line approach to provide improved protein sequence coverage toward protein identification in a combined top-down/bottom-up proteome platform.
Asunto(s)
Proteínas/aislamiento & purificación , Proteoma , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
A simple process for realizing stable and reliable electrospray ionization (ESI) tips in polymer microfluidic systems is described. The process is based on the addition of a thin hydrophobic membrane at the microchannel exit to constrain lateral dispersion of the Taylor cone formed during ESI. Using this approach, ESI chips are shown to exhibit well-defined Taylor cones at flow rates as low as 80 nL min(-1) through optical imaging. Furthermore, stable electrospray current has been measured for flow rates as low as 10 nL min(-1) over several hours of continuous operation. Characterization of the electrospray process by optical and electrical monitoring of fabricated ESI chips is reported, together with mass spectrometry validation using myoglobin as a model protein. The novel process offers the potential for low-cost, direct interfacing of disposable polymer microfluidic separation platforms to mass spectrometry.