Claire Smadja

Use of self assembled magnetic beads for on-chip protein digestion

The use of grafted trypsin magnetic beads in a microchip for performing protein digestion is described. The PDMS device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the center of the chip channel. This allows for the formation of a low-hydrodynamic resistance plug of magnetic trypsin beads that serves as a matrix for protein digestion. This device represents an inexpensive way of fabricating a multi open-tubular-like column with an appropriate pore size for proteins. Kinetics studies of the hydrolysis of a model peptide show a 100-fold increase in digestion speed obtained by the microsystem when compared to a batch wise system. This system also offers the great advantage of easy replacement, as the bead matrix is easily washed out and replaced. High performance and reproducibility for digesting recombinant human growth hormone are confirmed by analysing the digest products in both CE and MALDI-TOF MS. Similar sequence coverage (of about 44%) is obtained from MS analysis of products after 10 minutes on-chip and 4 h with soluble trypsin in bulk.

Controlled proteolysis of normal and pathological prion protein in a microfluidic chip.

A microreactor for proteinase K (PK)-mediated protein digestion was developed as a step towards the elaboration of a fully integrated microdevice for the detection of pathological prion protein (PrP). PK-grafted magnetic beads were immobilized inside a polydimethylsiloxane (PDMS) microchannel using a longitudinal magnetic field parallel to the flow direction and a magnetic field gradient, thereby forming a matrix for enzymatic digestion. This self-organization provided uniform pore sizes, a low flow resistance and a strong reaction efficiency due to a very thin diffusion layer. The microreactors performance was first evaluated using a model substrate, succinyl-ala-ala-ala-paranitroanilide (SAAAP). Reaction kinetics were typically accelerated a hundred-fold as compared to conventional batch reactions. Reproducibility was around 98% for on-chip experiments. This microsystem was then applied to the digestion of prion protein from brain tissues. Controlled proteolysis could be obtained by varying the on-chip flow rate, while a complete proteolysis of normal protein was achieved in only three minutes. Extracts from normal and pathological brain homogenates were finally compared and strong discrimination between normal and pathological samples was demonstrated.

On-chip tryptic digest with direct coupling to ESI-MS using magnetic particles

As a step toward a fully automated front‐end microfluidic chip for MS proteomics, we propose a system capable of performing online tryptic digest and ESI‐MS, using a replaceable on‐chip digestion microcolumn based on self‐assembled magnetic particles.

High performance liquid chromatography separation of structurally related enkephalins on quaternary ammonium-embedded stationary phase in isocratic mode

Separation of twelve enkephalins was investigated on a quaternary ammonium-embedded stationary phase (Stability BS-C23). Variation of buffer pH of the mobile phase highlighted the complex relationship between repulsive/attractive electrostatic interactions and the reversed-phase partitioning mechanism. The effect of three different anions employed as additives (phosphate, chloride and perchlorate) was examined at various concentrations and two pH values (acidic and neutral). At pH 2.5, an increase in the anion eluent concentration resulted in a higher retention factors of positively charged enkephalins. This effect was more pronounced when perchlorate ions were added to the mobile phase rather than phosphate and chloride ions, due to chaotropic and ion-pairing effects. In contrast, at pH 7.5, retention factors of negatively charged enkephalins decreased when these salts were added, due to an anion-exchange mechanism. Perchlorate caused a sharper decrease than chloride and phosphate anions did. The results presented here provide insight into the possible adjustment of retention and separation of peptides on a mixed-mode stationary phase (BS-C23) by a careful control of the buffer pH, the nature and concentration of anions, added to the buffer, and organic modifier content.

Monodisperse Carboxyl‐Functionalized Poly(Ethylene Glycol)‐Coated Magnetic Poly(Glycidyl Methacrylate) Microspheres: Application to the Immunocapture of β‐Amyloid Peptides

Identification and evaluation of small changes in β-amyloid peptide (Aβ) levels in cerebrospinal fluid is of crucial importance for early detection of Alzheimers disease. Microfluidic detection methods enable effective preconcentration of Aβ using magnetic microparticles coated with Aβ antibodies. Poly(glycidyl methacrylate) microspheres are coated with α-amino-ω-methoxy-PEG5000 /α-amino-ω-Boc-NH-PEG5000 Boc groups deprotected and NH2 succinylated to introduce carboxyl groups. Capillary electrophoresis with laser-induced fluorescence detection confirms the efficient capture of Aβ 1-40 peptides on the microspheres with immobilized monoclonal anti-Aβ 6E10. The capture specificity is confirmed by comparing Aβ 1-40 levels on the anti-IgG-immobilized particles used as a control.

Capillary electrophoresis for rapid identification of monoclonal antibodies for routine application in hospital

mAbs are widely used in cancer therapy. Their compounding, performed just before their administration to patients, is executed in a production unit of the hospital. Identification of these drugs, individually prepared in bags for infusion before patient administration, is of paramount importance to detect potential mistakes during compounding stage. A fast and reliable analytical method based on CZE combined to a cationic capillary coating (hexadimethrine bromide) was developed for identification of the most widely used compounded therapeutic for cancer therapy (bevacizumab, cetuximab, rituximab, and trastuzumab). Considering the high structural and physico‐chemical similarities of these mAbs, an extensive optimization of the BGE composition has been performed. The addition of perchlorate ions and polysorbate in the BGE greatly increased the resolution. To validate the method, an internal standard was used and the relative migration times (RTm) were estimated. Very satisfactory RSDs of the RTm for rituximab (0.76%), cetuximab (0.46%), bevacizumab (0.31%), and trastuzumab (0.60%) were obtained. The intraday and interday RSD of the method were less than 0.32 and 1.3%, respectively for RTm. Significant differences between theses RTms have been demonstrated allowing mAbs identification. Finally, accurate mAbs identification has been demonstrated by a blind test.

Hexylacrylate-based mixed-mode monolith, a stationary phase for the nano-HPLC separation of structurally related enkephalins

The potential of an in situ photopolymerized hexylacrylate-based monolithic stationary phase-bearing sulfonic acid groups was investigated by studying the chromatographic retention of small structurally related peptides (enkephalins) by nano-LC. Several retention mechanisms were highlighted. First, a reverse-phase chromatographic behavior toward neutral solutes due to hexylacrylate-moieties was demonstrated. Second, an evaluation of the influences of buffer pH suggested the involvement of a cation-exchange mechanism due to the presence of 2-acrylamido-2-methyl-1-propanesulfonic acid. This cation-exchange phenomenon was confirmed by the clear influence of Na+ concentration in the mobile phase on peptide retention.

Solid supports for extraction and preconcentration of proteins and peptides in microfluidic devices: A review

Determination of proteins and peptides is among the main challenges of todays bioanalytical chemistry. The application of microchip technology in this field is an exhaustively developed concept that aims to create integrated and fully automated analytical devices able to quantify or detect one or several proteins from a complex matrix. Selective extraction and preconcentration of targeted proteins and peptides especially from biological fluids is of the highest importance for a successful realization of these microsystems. Incorporation of solid structures or supports is a convenient solution employed to face these demands. This review presents a critical view on the latest achievements in sample processing techniques for protein determination using solid supports in microfluidics. The study covers the period from 2006 to 2015 and focuses mainly on the strategies based on microbeads, monolithic materials and membranes. Less common approaches are also briefly discussed. The reviewed literature suggests future trends which are discussed in the concluding remarks.

Quality Control of Therapeutic Monoclonal Antibodies at the Hospital After Their Compounding and Before Their Administration to Patients

Monoclonal antibodies (mAbs) are widely used in cancer therapy and recently many new mAbs have gained EMA and FDA approvals for oncology indications. Here we describe a highly reproducible CZE method, relying on a cationic coating allowing separation and identification of a complex mixture of four compounded mAbs widely used in cancer therapy (cetuximab, rituximab, bevacizumab, and trastuzumab).

Chemical engineering of self-assembled Alzheimer's peptide on a silanized silicon surface.

The aim of this work is to develop a sensitive and specific immune-sensing platform dedicated to the detection of potential biomarkers of Alzheimers disease (AD) in biological fluids. Accordingly, a controlled and adaptive surface functionalization of a silicon wafer with 7-octenyltrichlorosilane has been performed. The surface has extensively been characterized by atomic force microscopy (AFM; morphology) and X-ray photoelectron spectroscopy (XPS; chemical composition) and contact angle measurements. The wettability of the grafted chemical groups demonstrated the gradual trend from hydrophilic to hydrophobic surface during functionalization. XPS evidenced the presence of silanes on the surface after silanization, and even carboxylic groups as products from the oxidation step of the functionalization process. The characterization results permitted us to define an optimal protocol to reach a high-quality grafting yield. The issue of the quality of controlled chemical preparation on bioreceiving surfaces was also investigated by the recognition of one AD biomarker, the amyloid peptide Aβ 1-42. We have therefore evaluated the biological activity of the grafted anti Aβ antibodies onto this silanized surface by fluorescent microscopy. In conclusion, we have shown, both qualitatively and quantitatively, the uniformity of the optimized functionalization on slightly oxidized silicon surfaces, providing a reliable and chemically stable procedure to determine specific biomarkers of Alzheimer disease. This work opens the route to the integration of controlled immune-sensing applications on lab-on-chip systems.