Search

Laura Ceriotti,1 Paolo Buratti,1 Enrico Stefano Corazziari,2 Marisa Meloni1 1In vitro Innovation Center, VitroScreen srl, Milan, Italy; 2Department of Gastroenterology, IRCCS Humanitas Research Hospital, Rozzano (Milan), ItalyCorrespondence: Laura Ceriotti, VitroScreen, in vitro Innovation Center, Via Mosè Bianchi, 103, Milan, 20149, Italy, Email laura.ceriotti@vitroscreen.comPurpose: A novel experimental design based on a human-reconstructed oesophageal epithelium (HO2E) model has been applied to quantitively assess the properties of a set of liquid formulations, Device A (Gerdoff® Protection), Device B (Esoxx® One), and Device C (Marial® gel) developed to form a temporary physical barrier on the oesophageal epithelium and modify epithelial permeability so to protect the oesophageal mucosa from refluxate components.Methods: The formulations were applied to a prewetted HO2E model for 15 min. Then, a 0.5% caffeine solution was applied, and its penetration kinetics was assessed at 1 h and 2 h in acidic environments (pH= 3.3) to mirror exposure of the oesophageal mucosa to acidic reflux in GORD patients. Caffeine permeated into the basolateral compartment (evaluated by HPLC-UV) and Lucifer yellow (LY) permeability were quantified 15 min after application of the caffeine in acidic environments.Results: At the 15 min timepoint, Device A reduced caffeine permeation by 77.2% and LY flux by 30.4% compared to the untreated control and with a faster mode of action than that of the other liquid formulations. Transepithelial caffeine flux was reduced, albeit with different timing and efficiency, by all three compounds up to the end of the 2 hour experiment. At 1 h, Device A reduced the caffeine flux by 79.2%; Device B, by 67.2%; and Device C, by 37%.Conclusion: These results confirm the ability of the medical devices tested to interact with the oesophageal epithelium and create a temporary physical protective film for up to 2 hours after their application. The results underline differences in the mechanism of action of the three medical devices, with Device A performing faster than the other formulations. The overall results support the relevance of the reconstructed mucosal model to investigate oesophageal epithelium–product interactions and precisely differentiate liquid formulation performance.Keywords: gastro-oesophageal reflux disease, film-forming properties, caffeine permeation, epithelial permeability, epithelial protection, Lucifer yellow assay

Marisa Meloni, Paolo Buratti, Francesco Carriero, Laura Ceriotti VitroScreen, In Vitro Innovation Center, Milan, 20149, ItalyCorrespondence: Laura Ceriotti Via Mosè Bianchi, 103, Milan, 20149, ItalyTel +39 02 89077608Email laura.ceriotti@vitroscreen.comPurpose: A novel experimental model based on a 3D reconstructed human oesophageal epithelium model (HO2E) has been developed to investigate the structural and functional changes of the oesophageal epithelium following exposure to a solution of HCl 0.1 N (pH = 1.2) mirroring GERD microenvironment condition.Methods: The barrier structure modification after the exposure to the acid solution on HO2E tissues was investigated immediately after damage induction and after 1 hour post incubation and compared to HO2E tissues exposed to phosphate buffered saline solution. Immunofluorescence (IF) was applied to quantify the expression and localization of barrier function proteins: Claudin-1 (CLDN-1), Claudin-4 (CLDN-4), Zonulin-1 (ZO-1), E-Cadherin and Mucin-1 (MUC1). Barrier functionality was measured by TEER.Results: In the acidic microenvironment, TEER measurement has shown some limitations and results were not applicable, whereas the evaluation of protein localization and quantification provided clear and robust evidence of the damage which occurred to the epithelium barrier structure. CLDN-4 expression significantly decreased after exposure to acid. ZO-1 protein appeared upregulated immediately after exposure to HCl and was mainly localized in the cytoplasm and not on the cell membrane. This different localization was also observed for CLND-1. CLDN-1, MUC1 and, to a lower extent, ZO-1 expression increased during the post-incubation period.Conclusion: The relevant tissue biomarkers identified, CLDN-1 and MUC1, can be used to monitor TJ structure and epithelial barrier recovery after acid-induced damage which, in our experimental conditions, were non-destructive and suitable for recovery studies. The established model can be useful to investigate the mechanism of action of formulations acting on this specific pathophysiological condition and/or designed to potentiate the physiological defense mechanisms of oesophageal mucosa.Keywords: gastro-oesophageal reflux disease, GERD, 3D reconstructed human oesophageal epithelium, Claudin-1, Claudin-4, E-Cadherin, Zonulin-1, Mucin-1, TEER measurement

Francesca Rescigno, Laura Ceriotti, Marisa Meloni VitroScreen, In Vitro Innovation Center, Milan, ItalyCorrespondence: Francesca RescignoVitroScreen, In Vitro Innovation Center, via Mosè Bianchi, 103, Milan, 20149, ItalyEmail francesca.rescigno@vitroscreen.comObjective: Dermis spheroids from different donors (40 and 50 years old) were developed from primary fibroblasts to demonstrate their capacity to synthetize and organize the main dermal structural components when cultured in 3D microenvironment, forming endogenous de novo ECM according to their potential metabolic activity.Methods: Dermis spheroids were produced from primary human dermal fibroblasts at early passages in hanging drop culture system. Dermis models were characterized in terms of spheroid diameter, PICP release, collagen III and CD44 expression.Results: An increase of collagen III synthesis (101%) was found in the young donor compared to the old donor (23.5%) after seven days of culture by immunofluorescence. The progressive ECM assembly over the time and dermis maturation was showed by Masson’s trichrome staining and by immunofluorescence for collagen III and CD44; both molecules significantly accumulated in the dermal compartment from day seven to day 10 of culture with a global decrease for both spheroid models after 21 days of culture.Conclusion: Our results showed that specific culture conditions in the 3D scaffold-free microenvironment allowed the physiological and progressive ECM assembly of miniaturized dermis models reflecting phenotypic profile features of “young” and “old” native tissue from which cells were isolated with a potential application to personalized care approaches in dermatological research on aging processes and medicine.Keywords: 3D culture, dermis, scaffold-free spheroids, intrinsic aging, extracellular matrix, tissue remodeling

Gaia Pellegatta,1 Marco Spadaccini,1 Laura Lamonaca,1 Vincenzo Craviotto,1 Ferdinando D’Amico,1 Laura Ceriotti,2 Marisa Meloni,2 Alessandro Repici1 1Humanitas Clinical and Research Center and Humanitas University, Digestive Endoscopy Unit, Division of Gastroenterology, Rozzano, MI, Italy; 2VitroScreen, Milano, ItalyCorrespondence: Gaia PellegattaDigestive Endoscopy Unit, Division of Gastroenterology, Humanitas Research Hospital, Via Manzoni 56, Rozzano, MI 20089, ItalyTel +390282247091Email gaia.pellegatta@humanitas.itPurpose: New medical devices that contain hyaluronic acid (HA) and chondroitin sulphate (CS), with or without antacid components, have been developed for the treatment of gastroesophageal reflux disease (GERD) with the aim of improving oesophageal mucosal defences by creating a film on the oesophageal mucosa and acting as a mechanical barrier against the noxious components of refluxate, both acidic and basic.Methods: The film-forming and protective efficacy of medical device A based on HA and CS plus aluminium hydroxide, device B combining HA and CS with magnesium trisilicate and device C with only the combination of HA and CS was tested on a reconstructed human oesophageal epithelium (HO2E/S/5) as a biological model in 2 different pH environments, neutral and acidic, to mimic realistic conditions. Caffeine penetration kinetics and Lucifer yellow (LY) permeability modifications induced by these products were compared to those induced by a negative control series (saline solution, code NC) and positive control series (white Vaseline, code V) under neutral and acidic pH conditions.Results: Under neutral and acidic pH conditions, compared to the negative control, all the products tested reduced (> 80% and 85– 90%, respectively) the caffeine passage, and no significant difference was observed among the products tested. Under neutral and acidic conditions, the LY permeabilities registered with device A and device C were not different from that registered with the negative control, while an LY flux% increase was calculated after 2 hrs of treatment (21.1%) with device B under acidic conditions.Conclusion: These results confirm the ability of the products tested to interact with the oesophageal epithelium in order to adhere and create a stable protective film for at least 2 hours after their homogeneous distribution on the epithelium surface. Further clinical studies are needed to test these devices in the topical treatment of gastroesophageal reflux symptoms.Keywords: hyaluronic acid, chondroitin sulphate, gastroesophageal reflux disease, antacid, caffeine, Lucifer yellow

Laura Ceriotti,1 Silvia Balzaretti,1 Salvatore Barone,2 Marisa Meloni1 1VitroScreen, Milan, Italy; 2NTC S.r.l., Milan, ItalyCorrespondence: Laura CeriottiVitroScreen, Via Mosè Bianchi 103, Milan 20149, ItalyTel +39 02 89077608Email laura.ceriotti@vitroscreen.comObjective: To assess the eye tolerability of a buffered ophthalmic solution containing microglycine (sodium hydroxymethylglycinate, mwaterTM) in an in vitro model.Materials and Methods: A multiple endpoint analysis (MEA) approach was applied to the reconstructed human corneal epithelium (HCE) model. Sodium hydroxymethylglycinate solution (0.04%) and two ophthalmic ointments containing microglycine (Protectorial, containing 0.02% of sodium hydroxymethylglycinate, and Edenight, containing 0.04% of sodium hydroxymethylglycinate) were investigated. The buffered solution and the ointments were tested on HCE after acute (one application in 24 hrs, followed or not by 16 hrs of recovery) or repeated (one application per day for three consecutive days) exposures; benzalkonium chloride (BAK) 0.01% and saline isotonic solution were used as positive and negative controls, respectively. Cellular viability, trans-epithelial electrical resistance (TEER), lactate dehydrogenase (LDH) release and histo-morphology were evaluated.Results: BAK 0.01% toxicity in HCE was confirmed for the 24+16 hrs acute and repeated exposure protocols, while, after 24–hours acute treatment, only modifications of the superficial cell layer were visible compared with the negative control. Sodium hydroxymethylglycinate had a very good tolerability profile and a neutral impact on the corneal surface after acute or repeated exposure. The Protectorial and Edenight ointments preserved cell viability in the different exposure protocols, suggesting a good local tolerability profile. Modifications of the superficial layers were observed on histo-morphological analysis and confirmed by increased release of LDH after 24+16 hrs acute exposure (+65% and +76% for Protectorial and Edenight, respectively) and TEER values after 24+16 hrs and 72 hrs exposure protocols. These results were dependent on the ointments’ accumulation on the corneal epithelium due to their physical form (semi-solid) and lipophilic properties.Conclusion: Sodium hydroxymethylglycinate, alone or as part of eye ointments, was found to be non-toxic after acute or repeated exposure in the reconstructed HCE model.Keywords: BAK, biocompatibility, delayed toxicity, HCE, microglycine

Purpose: To develop an in vitro model of severe immunocompetent-dry eye disease (ic-DED) and to investigate the mechanism of action of a T-lysial ocular surface modulator., Materials and Methods: The reconstructed human corneal epithelium (HCE) was exposed to dryness stimuli. THP-1 cell infiltration into HCE was monitored at 4 h and 24 h from T-lysial application by immunohistochemistry (CD14, CD86, AQP3) and molecular biology (AQP3, TLR4 and TNF-α)., Results: A reduction of CD14, CD86 and AQP3 was observed after T-lysial treatment at 24 h. TLR4 was overexpressed in ic-DED model and downregulated by T-Lysial after 24 h. TNF-α expression was not modified., Conclusion: The ic-DED model can be used to monitor the migration and differentiation of THP-1 into HCE. T-lysial was found to exert anti-inflammatory activity. This experimental model is a promising tool to study the crosstalk between epithelial and immune cells, providing new insights on the mechanisms of DED onset.

Plant biomasses growing in bioreactor could be developed as production systems for cosmetic ingredients, nutraceuticals and food additives. We previously reported that the red carrot cell line R4G accumulates high levels of anthocyanins, which are potent antioxidants with multiple health-promoting properties. To investigate the industrial potential of this cell line in detail, we tested extract for antioxidant and anti-inflammatory activity in the mouse monocyte/macrophage cell-line J774A.1 and in reconstructed skin tissue models. We also compared the R4G extract to commercial carrot extracts in terms of stability and metabolomic profiles. We found that the R4G extract have potent antioxidant and anti-inflammatory activities, protecting mammalian cells from the oxidative stress triggered by exposure to bacterial lipopolysaccharides and H 2 O 2 . The extract also inhibited the nuclear translocation of NF-κB in an epidermal skin model, and induced the expression of VEGF-A to promote the microcirculation in a dermal microtissue model. The anthocyanins extracted from R4G cells were significantly more stable than those found in natural red carrot extracts. Finally, we showed that R4G extract has similar metabolomic profile of natural extracts by using a combination of targeted and untargeted metabolomics analysis, demonstrating the safety of R4G carrot cells for applications in the nutraceutical and food/feed industries., (Copyright © 2020 Bianconi, Ceriotti, Cuzzocrea, Esposito, Pressi, Sgaravatti, Bertaiola, Guarnerio, Barbieri, Semenzato, Negri, Commisso, Avesani and Guzzo.)

A multiple endpoint analysis (MEA) approach on human reconstructed corneal epithelium (HCE) model has been applied to assess the biocompatibility (cytotoxicity and irritation potential) of medical devices (MD): ophthalmology literature clearly shows the need to better assess these products to exclude any potential chronic damage to the ocular surface. Preserved eye drops (Artelac Multidose, Optive multidose and Artelac Rebalance Multidose) and the same without preservative (Artelac Edo, Optive Unidose, Artelac Rebalance Unidose) and Thealoz Duo were tested after acute (24 h + 16 h post incubation) and repeated (2 applications/day for 72 h) exposure using BAK 0.01% as positive control on HCE. Cellular viability, trans-epithelial electrical resistance measurements, LDH release and occludin gene expression were evaluated for each product to discriminate the potential toxicity of preservatives. The BAK 0.01% toxicity on HCE was confirmed following both exposures. The analysis of the same parameters reveals that the 72 h exposure was suitable to identify toxicity and damages to the ocular surface even for 'soft' preserved MD. The results confirm the reliability, sensitivity and predictivity of the MEA on HCE in detecting subclinical signs of cellular toxicity: 'soft' preservatives resulted toxics suggesting that delayed toxicity should be integral part of the biocompatibility assessment of ophthalmic formulations intended for long-term use., (Copyright © 2019. Published by Elsevier Inc.)

Two methods for protein patterning on antifouling surfaces have been applied to analyze the density and bioactivity of the proteins after deposition. Microcontact printing has been used as a technique to transfer fibronectin through conformal contact, while piezoelectric deposition has been employed as a non-contact technique for producing arrays of fibronectin (FN). Plasma deposited polyethylene oxide-like (PEO-like) films have been used as non-fouling background to achieve the bioadhesive/biorepellent surface contrast. Both patterning methods allow the direct fabrication of protein arrays on a non-fouling substrate, and the subsequent formation of a pattern of stem cells by cell attachment on the arrayed substrates. Microcontact printing produced fully packed homogeneous fibronectin patterns, much denser than microspotted patterns. Both printing and spotting technologies generated functional protein arrays, their bioactivity being primarily modulated by the density of the deposited protein layer. Optimization of the FN parameters used for deposition has lead to the achievement of high-quality microarrays with large population of neural stem cells immobilized in the patterns in serum-free conditions, where cells exhibit a more homogeneous starting population and factors influencing fate decisions can be more easily tracked. The immunorecognition of fibronectin targeted antibodies, as well as the cell density, increase with the protein density up to a saturation point. Over 100 ng/cm² of fibronectin on the surface leads to a decrease in the number of attached cells and a raise of cell spreading.

The last decade has witnessed a significant increase in interest in whole-cell biosensors for diverse applications, as well as a rapid and continuous expansion of array technologies. The combination of these two disciplines has yielded the notion of whole-cell array biosensors. We present a potential manifestation of this idea by describing the printing of a whole-cell bacterial bioreporters array. Exploiting natural bacterial tendency to adhere to positively charged abiotic surfaces, we describe immobilization and patterning of bacterial "spots" in the nanolitre volume range by a non-contact robotic printer. We show that the printed Escherichia coli-based sensor bacteria are immobilized on the surface, and retain their viability and biosensing activity for at least 2 months when kept at 4 °C. Immobilization efficiency was improved by manipulating the bacterial genetics (overproducing curli protein), the growth and the printing media (osmotic stress and osmoprotectants) and by a chemical modification of the inanimate surface (self-assembled layers of 3-aminopropyl-triethoxysilane). We suggest that the methodology presented herein may be applicable to the manufacturing of whole-cell sensor arrays for diverse high throughput applications.

The interaction of biological systems with synthetic material surfaces is an important issue for many biological applications such as implanted devices, tissue engineering, cell-based sensors and assays, and more generally biologic studies performed ex vivo. To ensure reliable outcomes, the main challenge resides in the ability to design and develop surfaces or artificial micro-environment that mimic 'natural environment' in interacting with biomolecules and cells without altering their function and phenotype. At this effect, microfabrication, surface chemistry and material science play a pivotal role in the design of advanced in-vitro systems for cell culture applications. In this chapter, we discuss and describe different techniques enabling the control of cell-surface interactions, including the description of some techniques for immobilization of ligands for controlling cell-surface interactions and some methodologies for the creation of well confined cell rich areas.

"Macromolecules to PDMS transfer" technique relying on the direct entrapment of macromolecules spots during PDMS polymerisation is proposed as an alternative for the easy and simple PDMS surface modification. In the present work, the development of three different applications based on this procedure is presented as proof of the method potentialities. First, C-reactive protein (CRP) sandwich immunoassay using immobilised monoclonal anti-CRP antibodies was developed for sepsis diagnosis. The preserved integrity of the immobilised monoclonal immunoglobulin permitted the sensitive detection of free CRP in human sera (LOD=12.5 microg/L, detection ranging over two decades). Then, rheumatoid arthritis diagnosis through the rheumatoid factor (RF) detection based on rabbit immunoglobulins immobilisation allowed the detection of specific antibodies in human sera samples down to low RF levels (detection range 5.3-485 IU/mL). Finally, the "Macromolecules to PDMS transfer" procedure was used to easily and rapidly produce fibronectin-based cell culture arrays. The successful attachment of HeLa and BALB/3T3 cells was demonstrated with optical microscopy and specific staining of actin and vinculin.

Bio-functionalized surfaces were prepared to study the adherence and differentiation capacity of neural stem cells derived from human umbilical cord blood (HUCB-NSC). Cell growth platforms containing arranged arrays of adhesive molecules were created by microcontact printing on a biologically inert surface. Biomolecules used to prepare microarray platforms included the extracellular matrix protein fibronectin and the polyaminoacid poly-L-lysine. HUCB-NSC plated on microplatforms at various serum conditions showed serum and molecule type dependent capacity for adhesion and differentiation. Poly-L-lysine allowed the maintenance of stem-like non differentiated cells attached to the surface, whereas fibronectin promoted spreading and neural commitment. Serum deprivation did not influence the attachment of HUCB-NSC to fibronectin, but significantly enhanced the attachment to poly-L-lysine and promoted dBcAMP induced neuronal differentiation. A bio-pattern of squares with interconnecting lines was used to guide neuronal differentiation by directing cell protrusion outgrowth. Tailoring the geometry of the bio-pattern enabled directing and monitoring of the neural stem cells. development in the large scale multiparameter biotests.

We present a method for patterning neural stem cells based on pre-patterning polypeptides on a cell-repellent surface (poly(ethylene) oxide-like, PEO-like, plasma-deposited films). The method ensures cell attachment and stability for several weeks, as well as it allows cell migration and differentiation. Various patterns of approximately 1 nm thick cell adhesive poly-L-lysine (PLL) have been created on a cell-repellent PEO-like matrix by microcontact printing using different array configurations and printing conditions. The cell-repellent property of PEO-like film determined the confinement of the cells on the printed patterns. Optimization of the printing method showed that the most homogeneous patterns over large areas were obtained using PLL diluted in carbonate buffer (100mM) at pH 8.4. Neural stem cells cultured on the PLL patterns in low serum and in differentiating medium over 20 days exhibited a good confinement to the polypeptide domains. The number of cells attached increased linearly with the micro-stamped PLL area. The cells were able to extend random axon-like projections to the outside of the patterns and presented high amount of ramifications when cultured in differentiating medium. Migration and axon-like outgrowth have been successfully guided by means of an interconnected squares configuration. The surfaces are suitable for controlling the patterning of stem cells and provide a platform for the assessment of the way how different cell arrangements and culture conditions influence cell interactions and cell developmental processes.

This paper describes a simple and convenient method to monitor on-line cell adhesion by electrical impedance measurements. Immortalized mouse fibroblasts, BALB/3T3, were cultured onto interdigitated electrode structures integrated into the bottom of an in-house fabricated device. Impedance modulus, phase, real and imaginary parts were considered separately and plotted as function of frequency and time to better understand and select the component giving more information on cell adhesion changes. For cytotoxicity assessment, the cells were treated with different concentrations of sodium arsenite used as model toxicant and their responses were monitored on-line. The half inhibition concentration, the required concentration to achieve 50% inhibition, derived from the measurements fall between the results obtained using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test and colony forming efficiency assay confirming the good sensitivity of the system. In term of impedance signal, the modulus results was found to be the most sensitive of the considered components for cytotoxicity testing of chemicals.

This work presents a method for the fabrication of well defined chemically active nano-patterned surfaces. Electron-beam lithography has been combined with plasma-based processes in order to create sub-micron carboxylic functional areas over a non-bio-adhesive matrix. Characterization of the patterned surface by several surface analysis techniques reveals that this patterning technique is compatible with the plasma polymerization process in order to fabricate chemically active features with lateral size down to 300 nm. Moreover, experiments with a model protein (bovine serum albumin) on the patterned surfaces show preferential adhesion on the active region indicating the ability of this method for the design of biosensing platforms.

The recent European Commission REACH (Registration, Evaluation and Authorisation of Chemicals) policy outlines a plan for toxicological testing by using alternative non-animal in vitro methods. In this context, there is a need to develop and standardise high-throughput screening (HTS) methods for studying the cytotoxicity induced by chemicals. Electrochemical impedance spectroscopy (EIS) can be considered as a complementary technique to alternative in vitro testing for studying cell adhesion to the substrate, and can give real-time and kinetic information on cell responses to a toxicant. This paper describes the development of a home-made chip based on impedance spectroscopy, and its application in studying the kinetics of BALB/3T3 cell adhesion and the cellular responses to a toxic product as a function of time. Concentrations of sodium arsenite, ranging from 10 microM up to 1000 microM, were tested in the system, and the results were compared with those obtained with standard protocols used to study basal cytotoxicity induced by chemicals in the BALB/3T3 cell line. The results show that the sensitivity of the developed chip was better than that with the MTT test, with the additional advantages of online monitoring.

Due to the mounting evidence for altered lipoprotein and cholesterol-lipoprotein content in several disease states, there has been an increasing interest in analytical methods for lipoprotein profiling for diagnosis. The separation of low- and high-density lipoproteins (LDL and HDL, respectively) has been recently demonstrated using a microchip capillary electrophoresis (CE) system [1]. In contrast to this previous study, the present report demonstrates that LDL analysis can be performed in an uncoated glass microchannel. Moreover, by adding sodium dodecyl sulfate (SDS) to the sample at a concentration well below the critical micellar concentration prior to injection, the LDL peak undergoes a focusing effect and exhibits an apparent efficiency of 2.2 x 10(7) plates/m. Laser light scattering experiments demonstrate that the low concentration of SDS used does not significantly alter lipoprotein particle size distribution within the time course that the analysis is performed. It is thus hypothesized that SDS nondisruptively coats LDL particles. The peak sharpening effect, observed only when SDS is added solely to the sample, is probably due to a mobility gradient created between the sample and the running buffer. The chip-based method demonstrated here has the potential for rapid analysis and sensitive detection of different LDL forms of clinical relevance.

The development of a new assay for lipoproteins by capillary electrophoresis in fused-silica capillaries and in glass microdevices is described in this paper. The separation of low-density (LDL) and high-density (HDL) lipoproteins by capillary zone electrophoresis is demonstrated in fused-silica capillaries with both UV absorption and laser-induced fluorescence detection. This separation was accomplished using Tricine buffer (pH 9.0) with methylglucamine added as a dynamic coating. With UV detection, LDL eluted as a relatively sharp peak with a migration time of approximately 11 min and HDL eluted as a broad peak with a migration time of 12.5 min. Fluorescence detection of lipoproteins stained with NBD-ceramide was used with the same buffer system to give comparable results. Furthermore, fluorescence staining of human serum samples yielded results similar to the fluorescently stained LDL and HDL fractions, showing that this method can be used to quantify lipoproteins in serum samples. The method was also used to detect lipoproteins in glass micro-CE devices. Very similar results were obtained in microdevices although with much faster analysis times, LDL eluted as a sharp peak at approximately 25 s and HDL as a broad peak at slightly longer time. In addition, higher resolution was obtained on chips. To our knowledge, these results show the first separation and detection of lipoproteins in a microfluidic device using native serum samples. Atomic force microscopy was used to characterize the rms surface roughness (Rq) of microfluidic channels directly. Devices with different surface roughness values were fabricated using two different etchants for Pyrex wafers with a polysilicon masking layer. Using 49% HF, the measured roughness is Rq = 10.9 +/- 1.6 nm and with buffered HF (NH4F + HF) the roughness is Rq = 2.4 +/- 0.7 nm. At this level of surface roughness, there is no observable effect on the performance of the devices for this lipoprotein separation.

A new polymer device for use with conventional particulate stationary phases for on-chip, fritless, capillary electrochromatography (CEC) has been realized. The structure includes an injector and a tapered column in which the particles of the stationary phase are retained and stabilized. The chips were easily fabricated in poly(dimethylsiloxane) using deep-reactive-ion-etched silicon masters, and tested using a capillary electrophoretic separation of FITC-labeled amino acids. To perform CEC, the separation channel was packed using a vacuum with 3-microm, octadecylsilanized silica microspheres. The packing was stabilized in the column by a thermal treatment, and its stability and quality were evaluated using in-column indirect fluorescence detection. The effects of voltage on electro-osmotic flow and on efficiency were investigated, and the separation of two neutral compounds was achieved in less than 15 s.

New acrylic polymers bearing oxirane groups were synthesized to be used in the production of coated capillaries. A fully automated coating procedure was devised based on the use of diluted water solutions of these polymers. The whole procedure required less than 30 min. The new polymers rapidly adsorbed from water onto the capillary wall, thus suppressing electroosmotic flow (EOF) to a negligible value. The adsorbed coatings were stable for hundreds of hours at high pH, temperature, and in the presence of 8 M urea. Efficient separations of acidic and basic proteins were achieved in the new phases.