Karine ; Faure
email: karine.faure@univ-lyon1.fr

Liquid Chromatography on chip

Keywords: chromatography, microsystem, electrochromatography, monolith, microfluidic, stationary phase, instrumentation

Liquid chromatography is one of the most powerful separation techniques as illustrated by its leading role in analytical sciences through both academic and industrial communities. Its implementation in microsystems appears to be crucial in the development of µTAS. If electrophoretic techniques have been widely used in miniaturized devices, liquid chromatography has faced multiple challenges in the downsizing process.

During the past five years significant breakthroughs have been achieved in this research area, in both conception and use of liquid chromatography on chip. This review emphasizes on the development of novel stationary phases and their implementation in microchannels. Recent instrumental advances are also presented, highlighting the various driving forces (pressure, electrical field) that have been selected and their respective ranges of applications.

I. Introduction

The beginning of the 21st century has witnessed explosive growth of chip-based analytical systems. This is no coincidence based on craze for miniaturized widgets, but the result of the attractive features chip-based analytical systems exhibit. Separation technique has been one of the first operations taking benefits from "Lab-on-a-Chip" conceptualization. As early as 1970, Terry and co-workers developed a gas chromatography on a silicon wafer [1]. When downsizing conventional separation systems to the micron scale, many improvements are observed in terms of separation performances, instrument versatility and effective costs. In most techniques, analysis times are reduced, efficiencies are improved. The integration of injectors minimizes the injected volumes to picoliters, which benefits to both sample consumption and band broadening. The zero-dead volume interconnections to detection systems contribute as well to this gain in efficiency. Moreover, microfabrication techniques allow the integration of multiple operations (sample preparation, microreactions) and the creation of multiple or parallel analytical devices, reducing the cost of the whole analytical chain. Mass production of these miniaturized devices and the extended use of thermoplastics participate at the reduction of costs and therefore encourage the development of disposable analytical microsystems, providing a suitable platform for samples that generate cross-contamination in conventional instruments.

Electrophoretic techniques (CZE, MEKC, IEF, ITP) are perfectly suited for miniaturization owing to the smaller separation lengths and resulting increase in electrical fields. This suitability is illustrated by the tremendous widespread of electrophoretic applications on chip, especially in the biochemistry and proteomic areas which are mostly dealing with charged solutes. However, there is a major need for additional selectivity on charged solutes and a definite necessity of solutions for the separation of neutral compounds.

Liquid chromatography is by far the most widely spread separation technique in conventional format.

The separation mechanism provided by the interaction of solutes with a stationary phase is a powerful, robust, yet versatile answer to the analysis of a wide range of compounds. Chip-based liquid chromatography equally benefits from the features that have done the success of electrophoresis on chip. Surprisingly, compared to the large diffusion of electrophoretic techniques in application areas such as biochemistry, very few groups are actually working on chromatography at microscale level. This delay is owed to the technical challenges that scientists are facing to introduce efficient stationary phases in microchannels. In the first part of this review, I will present the recent advances that have been reported in the field of novel chromatographic stationary phases at the microscale level. In the development of chromatography on chip, another interesting point has come into sight. The question of the driving force involved in the implementation of chromatography in Lab-on-a-chip devices is a great concern. Two strategies are considered: a "top-down" method consists in the miniaturization of conventional liquid chromatography instruments and will therefore use pressure as the liquid driving force, while the "bottom-up" idea consists in the implementation of a stationary phase in pre-existing miniaturized separation techniques and therefore employs electroosmosis mechanism to bring the fluids into movement.

A semantic issue arise from these two approaches. In the literature, a multitude of names have been given to chip-based separation techniques, leading to certain confusion. We will use throughout this text the prefix "Chip-"to confine the technique to miniaturized devices and to avoid any misunderstanding. Chip-LC will be restricted to pressure-driven liquid chromatography, while Chip-EC relates to chip-based electroosmosis-driven chromatography, i.e. electrochromatography.

If Chip-LC has already emerged on the commercial market, Chip-EC has been the most popular technique so far in research labs. The decision between these two techniques is usually taken in regards to personal scientific culture, whether chromatography on chip is regarded as a top-down or bottom-up strategy. In the second part of this review, I will try to point out the complementary character of these two approaches and the technological issues that arise from them.

II. A brief overview of stationary phases in chips

The aim of this section is to cover the time span of the last five years while focusing on the emergence of novel stationary phases on chip. The simplest way to integrate a stationary phase (and the first performed in chips) is to coat the channel walls with interacting solid phase, to realize open-channel chromatography. Increasing the available wall surface can be done by microstructuration conducting to the fabrication of pillar-type structures. A more conventional preparation of stationary phase is by complete filling of microchannels with externally prepared material such as silica particles and I will show some recent contributions in this field. Finally, the stationary phase can be created in-situ in the microchannels and I will illustrate the different materials that have been chosen for this purpose.

Open-channel chromatography

The very first LC -dedicated microsystem was fabricated in 1990 by Manz et al. [2], some 20 years ago. Based on Terry's GC-chip design, it involved a silicon wafer chemically etched to provide a 2µm

x 6 µm x 15 cm column hermetically sealed with a glass wafer. This work reflected the suitability of microsystems to open-channel chromatography. Reduction of the inner dimensions of the channels to 10 µm, ease of fabrication and low pressure drop were arguments in favour of the miniaturization of this separation technique. Although the very first open-channel chromatography actual separation was performed some 15 years ago, when Jacobson and co-workers coated a glass chip with octadecylsilane [3], for the successful separation of dyes with theoretical plate heights down to 4.5 µm, this technique has not widely spread in chip format. From 1994 to 2005, very little work has been carried out in this area, as illustrated in Pumera's review [4] who reported only 12 papers in 11 years, mainly performed by only four research groups [5][6][7][8]. Since 2005, coating of microsystems has been performed using many techniques such as sol gel [9] or nanoparticles deposition [10], but most of these surface treatments were used to reduce the adsorption character of microsystem bulk material, which contribution is especially detrimental when analysing biological solutes such as proteins. Very few strategies have been brought up to actually perform a selective adsorption or partitioning onto the wall surface for open-channel chromatography.

Functionalization of glass chips with C 8 -tetraethylorthosilicate (C 8 -TEOS) has been performed [11] following a previously reported procedure [6]. On the resulting open-channel Chip-LC, the authors demonstrate the reversed phase behavior of the coated stationary phase through separation of phenol and methylphenol. The paper focuses on the issue of continuous sampling and introduces a pressuredriven gated injection scheme. The main restriction of open-channel chromatography is the limited specific surface. The phase ratio (ratio between volume of stationary phase and volume of mobile phase) can be increased by generating a thick 3D coating. An illustration of this strategy is brought by Zeng [12]. PDMS chip was coated with hydrophilic polyacrylamide copolymerized with allyl-β-cyclodextrins. The resulting 3Dstationary phase allowed the discrimination of a mixture of enantiomeric FITC-labelled dansyl-valine and dansyl-aspartic acid within 100 s.

Another way to increase phase ratio can be performed by coating channel walls with particles.

Reversed-phase particles were embedded in a porous polymethacrylate stationary phase, as shown in Figure 1 for the successful separation of coumarins by shear-driven chip chromatography [13].

Gold nanoparticles have also been used in a multilayered coating procedure [14]. After functionalization of the glass channel with (3-mercaptopropyl)-trimethoxysilane, the resulting thiol groups linked BSA-gold nanoparticle complexes onto the surface. Immobilization of gold nanoparticles increase the amount of linked BSA, therefore increasing the capacity of the resulting stationary phase.

In open channel electrochromatography, efficiency is nearly as elevated as in zone electrophoresis, since only molecular diffusion has to be taken into account in band broadening. To counteract limited mass transfer, open-channel chromatography requires small channel dimensions, typically 10-20 µm, which is provided by microfabrication technologies. This technique is therefore highly suited for implementation in Lab-on-a-chip design. However, the reduction in specific surface drastically limits sample capacity and therefore the range of applications.

COMOSS, pillars and nanotubes

Microfabrication of pillars is an elegant strategy, taking advantages of microfabrication technology to combine small channel dimensions and large interacting surface [15]. Microfabricated columns, that are extremely difficult to create on the macroscale, present many advantages on a chromatographic point of view. The fabricated structures are inherently more regular that randomly packed particles, order that has a direct influence on eddy-diffusion. Theoretically, properly dimensioned pillars should hence result in higher efficiency [16].

In 1998, Regnier's group [17,18] introduced COMOSS (collocated monolith support structures), which is based on the direct fabrication of pillars during the microsystem fabrication process (Fig 2).

While the very first COMOSS were etched in quartz devices, it appears that very narrow channels induce frequent clogging, especially during post-fabrication functionalization and therefore discarding the chips was frequent. Quartz became too expensive and plastic devices were considered. PDMS COMOSS were fabricated as cheap, disposable microsystems with channels of 5 µm x 10 µm, ideal dimensions for open-channel chromatography [19]. To increase stability and selectivity, it was demonstrated that the introduction of an organic stationary phase on PDMS walls was compulsory. C 8 -and C 18 -moieties were introduced by silane modification procedure to improve peptides separation. Negative surfactants were then required in the mobile phase to generate sufficient electroosmotic mobility.

Another approach was developed the following year by Regnier's group to achieve surface modification of PDMS COMOSS with simultaneously charged and hydrophobic groups [20]. Radical polymerization was achieved on the PDMS pillars using Ce(IV) as a radical initiator. Mixed stationary phases such as acrylamidomethylpropylsulfonic acid-C 18 , poly (styrenesulfonic acid), poly (vinylsulfonic acid) or poly (acrylic acid) were investigated to provide both reversed phase interaction and electroosmotic flow. Efficiencies reached 600 000 plates/meter with analysis time around 5 min. PDMS, however, may not appear to be the best material option, due to its tendency to swell in hydroorganic solvents and to absorb hydrophobic solutes.

Cyclic olefin copolymer (COC) has demonstrated many positive features in terms of microfabrication, optical properties and chemical inertness. Using Regnier's design [17] to nanoimprint COC microsystems, Gustafsson et al [21] 7 material can generate an electroosmotic flow reaching a value of 4 x 10 -4 cm².V -1 .s -1 , but it exhibits reversed-phase behavior towards neutral analytes. At 20 % ACN in the mobile phase, three fluorescently labelled amines were separated according to their respective hydrophobicity. Observed plate heights reached the exceptional level of 0.5 µm for unretained compound due to electrokinetic stacking effect. Plate heights of 3.4 µm and 22 µm were achieved for Atto-532-hexylamine and Atto-532-octylamine, respectively, while the retention factors remained low (k = 0.4 and 1.5, respectively), mainly due to low carbon loading on the accessible surface.

Pressure-driven chromatography has also been carried out on such COC pillars design [22]. Again, separation of coumarins was achieved with high efficiencies (plate height around 5 µm for retained coumarin, see Fig 4) while retention factors remained low (k = 0.5 for coumarin C480 at 70/30 (v/v) water/methanol).

On the positive side, COMOSS fabricated structures lead to high efficiencies due to the reduction of eddy-diffusion. The microfabrication technology allows close-to-perfect column-to-column reproducibility and full control of pillar density. On the other hand, the use of non-porous pillars generates low phase ratio (in comparison with fully-filled channels) and therefore low sample capacity, limiting the analysis of samples with a wide range of concentrations. This remains the main limitation of native material as stationary phase.

As an answer to this issue, porous pillars have been etched in silicon chips [23]. A 550 nm porous layer was generated by anodisation with mesopores ranging between 5 and 15 nm, as shown in figure 5. This porous layer had no major influence on efficiency, but the authors calculated a 200-fold increase in the exchange surface, which could provide an answer to limited sample capacity.

A different approach regarding the same issues was considered in 2008 by Fonverne et al. [24]. The increase in phase ratio on ordered microfabricated pillars was obtained by growing carbon nanotubes on their surface. COMOSS were etched in a silicon wafer using DRIE etching process and in-situ growth of nanotubes was carried out before sealing the microsystem with Pyrex. Homogeneous layer of carbon nanotubes (30 nm diameter, 0.5-1 µm long) were obtained on silicon pillars (figure 6). Not only carbon nanotubes increased specific surface of COMOSS and hence capacity, but it also proved to be more retentive than C 18 -modified pillars towards polar solutes.

Pushing pillar strategy to a further extent, Goswami et al. [25] and Wu et al. [26] recently grew vertically-aligned carbon nanotubes directly in the microchannel as filling stationary phase (figure 7).

If the first group demonstrated proof of concept with the separation of tryptophan-tryptophan from unretained compound on native carbon nanotubes, Wu and co-authors [26] showed that the functionalization of carbon nanotubes, either with carboxylic groups or with 1-pyrenedodecanoic acid

Packed Particles

The transfer of liquid chromatography onto chip format can also be addressed as a simple miniaturisation challenge. Taking advantages of all the knowledge on silica particles that have traditionally been employed in LC columns as stationary phase is an attractive option, as the catalogue of available surface chemistry is wide and the expected chromatographic performances of such particles are well known. Two major issues can arise from the packing technology: reproducibility of the packing process and immobilization of particles. Three different strategies have been explored to pack particles in microchannel for chromatographic purposes.

The first approach considers particles packing in microchip as the direct continuation of the miniaturization trend that has undergone chromatographic column, consisting in the definition of a trapping zone using weirs [27][28][29]. Between 2000 and 2005, proofs of concept were achieved but the resulting chromatographic beds were fairly short due to the difficulty of packing process. On this path, Agilent HPLC-Chip has recently aroused most interest in the separation science community [30].

Made of polyimide, this chip is packed with slurries of conventional silica particles under 400 bars and with ultrasound assistance. Thanks to the wide range of particles available in Agilent catalogue (C 8 -,C 18 -and TiO 2 functionalized silica particles, various porosities), the number of application of the HPLC-Chip has dramatically increased in the literature. Not only this commercially-available system meets a large public avid of highly reliable micro-LC devices, but it also allows a better understanding of the influence of packing parameters on peak efficiency, such as channel geometry [31], particles size and packing procedure [32,33]. Another commercially available chip packed with silica particles, called cHiPLC, has recently been brought up to the market by Eksigent [34]. Based on glass material, the chip is presented on a docking station that allows its connection to any compatible detector and offer C18-functionalized silica particles of various sizes.

In a recent publication [33], Ehlert et al. assert that the gain of efficiency noticed in the HPLC-Chip compared to nano-LC is due to the fact that micromachined frits retaining the particulate bed in the microchip channel appeared to generate less dispersion than sintered frits. Reduction or elimination of frits has been regarded as one of the many challenges in the setup of stationary phase in microchannels. This idea of fritless chromatographic beds in microchips was already explored in the work by Ceriotti et al. [35] who defined tapered chromatographic column as to provoke a keystone for unretained compound and 120 000 plates/m for retained compounds, the stabilisation of the bed appeared to be somehow uncertain and required a thermal treatment. Following this path of fritless particle immobilization, Gomez's group [36,37] exploited the elasticity of PDMS material to entrap particles. The authors notice that during packing process executed at an intermittent pressure of 2 bars, the wall of PDMS channels would deform in an elastic way. The release of pressure induces a constraint on the packed particles that results in clamping of the whole chromatographic bed. Particles adjacent to PDMS walls are partially embedded in the elastomeric bulk of the microchip material. If particles seemed to be properly anchored, once again, a thermal treatment was necessary to assure the stability of the bed during chromatographic process. Evaluation of the chromatographic performances of such beds is currently under investigation.

In the search of the ideal packing procedure in microchannels, the difficulty stands in the production of a homogeneous bed. Packing of conventional particles has been achieved by low pressure in PDMS chips [27,[35][36][37], by high pressure in the HPLC-Chip [33] or even by electroosmotic flow [28,29].

One can also take advantage of surface-tension capillary forces resulting from a proper design of the microfluidic components to gain self -assembly of silica particles.

Recently, the use of self-assembly colloidal silica beads as the packing material has emerged from two groups. In Wirth's group [38], a 20 µm thick colloidal crystal of silica particles was obtained by evaporation and subsequent self-assembly of 200 nm calcinated silica colloids. After C 20 functionalization, the phase was used for the electrochromatographic reserved-phase separation of three dyes having similar electrophoretic mobilities. The authors demonstrated that, as expected, the high uniformity of packing generated by the crystallisation process induces a decrease of the A-term (eddy-diffusion) in the Van Deemter representation, reaching 2.5 µm, while the sub-micron size of the nanoparticles results in a much faster mass transport and therefore a low C-term of 1.2 ms. The selfassembly packing process was carried out on a quartz plate and subsequently enclosed by a PDMS cover, which led to a small volume of chromatographic phase.

Park et al. [39] develop the same self-assembly packing technique to fill a microstructured open channel with dimensions 100 µm x 80 µm x 25 mm, with 800 nm colloidal silica nanoparticles, as shown in figure 8. This considerably increased the volume of stationary phase. Unfortunately, the particles were not derivatized and only electrophoretic separation was carried out in this column.

Monoliths and continuous structures

Monoliths have been extensively studied as stationary phase in miniaturized format such as capillaries and microchips. Their synthesis, based on the polymerization of monomers in the presence of porogen, Deleted: The overall efficiency reached 2.5 µm, with a C-term of 1.2 ms. can easily be conducted in-situ and the resulting continuous bed exhibits a bimodal structure with large through-pores for flow and small pores for retention. The chromatographic characteristics can either be tuned before synthesis by adjusting the prepolymer composition, or modified in-situ by functionalization of the resulting bed. Many reviews are available, focusing on the study of monoliths in capillary format [40,41]. The size of the microfluidic channel being relatively close to capillary internal diameter, the transfer of monolith synthesis towards microchip is in theory quite straightforward. The very first syntheses of organic monoliths for EC purposes were carried out in glass chips [42][43][44][45], which material is similar to fused-silica capillaries. As in capillary, the silica walls were pre-treated with an adhesion promoter and then the channel was filled with the acrylate/methacrylate prepolymer before chemically- [42] or photo-initiated polymerization [43][44][45].

Since 2005, the technical issues for the synthesis in glass chips seem to have been answered and the work in this kind of chips has taken a step forward and is now focusing on specific issues, such as the integration of a extraction/preconcentration step before separation [46][47][48] or the establishment of a linear gradient elution in electrochromatography technique [49].

The growing market of chips fabricated in synthetic polymers reflects the enthusiasm towards a cheap, disposable, versatile device. However, special issues have to be faced up when implementing organic monoliths in plastic devices. The most widely spread polymeric material for cheap microfluidic application is polydimethylsiloxane (PDMS). The implementation of monoliths in this material was particularly difficult, as absorption of monomers and porogen into PDMS was recurrent.

Zeng et al. [12,50] prepared the γor β-CD bonded polyacrylamide monolithic column on a PDMS microfluidic device after surface functionalization using (trimethoxysilyl)-propyl methacrylate. In this case, crosslinker allyl-γ-or allyl-β-CD played as a chiral selector for enantioseparation of FITClabeled Dns-AAs. Covalent coating with a first layer of acrylate polymer was found to limit adsorption and subsequent photopolymerization of organic acrylate monolith was attainable [51].

Besides PDMS, other plastic materials were found to be less problematic in terms of absorption.

Polyimide, which was chosen by Agilent as the material for their commercial HPLC-Chips, is chemically inert. Its non-transparency forbids UV polymerization, but thermal polymerization was successfully achieved in this chip. Various monoliths have been synthesized, such as lauryl methacrylate, polystyrene-divinylbenzene [52] or methylstyrene-bisvinylphenyl ethane [53].

Cyclic olefin copolymer (COC) is another material of interest. Indeed, the soundness of this material eases its machining and enhances its durability. Furthermore, its transparency over a wide range of wavelength and its inertness towards most organic solvents, such as acetone, seems to offer a promising future to this material in term of analytical systems design. UV polymerization can be carried out in COC microdevices, which allow photopatterning and hence localization of the stationary phase in dedicated areas. One of the specifications of monolith synthesis in COC microchannels is the total exclusion of water in the polymerization mixture. Indeed, no polymerization is observed in COC Deleted: and I will here discuss only the recent advances that have been carried out in microchip format since 2005. Knapp's group has developed a UV-initiated synthesis of organic monolith in COC microchip to achieve the pressure-driven reversed-phase chromatography separation of proteins [54,55]. This monolith is prepared from a mixture of ethylhexyl methacrylate and EDMA as monomers and 1decanol as porogen. Functionalization of the COC channels with benzophenone and trimethylolpropane triacrylate (TMPTA) was required to firmly anchor the monolith to the channel wall, as advised by Stachowiak et al. [56], to avoid the formation of voids at the monolith-wall interface and to prevent movement of the monolith under high pressure. The resulting chip was then applied on the interface with a MALDI-TOF and an ESI mass spectrometry system for analysis of peptides mixtures. Peptide separation was achieved in Chip-LC-MS on this device but no chromatographic performances such as efficiency or retention factors are given.

F

Recently, Faure et al. synthesized a lauryl methacrylate-based monolith by UV initiation in COC microchip [57]. The porogenic system was a mixture of 1-propanol and 1,4-butanediol and no surface modification was performed before the introduction of the monolith prepolymer. The voids that may occur between monolith and channel walls were not detrimental to the separation of neutral compounds in electrochromatography mode. Efficiency reached 85 000 plates/meter. DeVoe's group reported a COC microdevice filled with polymethacrylate monolith for Chip-LC with a conventional LC gradient pumping system [48]. A 15 cm long monolith was formed in a serpentine channel to increase peak capacity. The plastic device and needle interfacing connections could withstand the outstanding pressure of 238 bar and 400 bar, respectively. The ability to locally synthesize monoliths by UV initiation enabled the definition of two zones for sample cleanup/ enrichment and chromatographic separation, respectively. This setup allows the separation of FITClabelled peptide by gradient elution.

Inorganic monoliths, such as silica monoliths, are prepared via a sol gel reaction of an alkoxysilane solution. In glass chip, no surface pretreatment is required since the silanols of the microfluidic channel walls are directly incorporated in the sol gel reaction. The chromatographic properties of silica monoliths are simply tuned since the skeleton size dictating permeability and the microporous structure dictating efficiency can be independently controlled. Another positive point in the use of silica monoliths as stationary phase in chromatography is their non-swelling behavior towards organic solvents, which may happen with some organic monoliths. One major drawback that has refrained the use of silica monolith in Lab-on-Chip strategies is the difficulty associated with their localization in precise zone. On the contrary of photopolymerized organic monoliths, silica sols must be confined in areas before gelation occurs.

To circumvent this problem, Morishima introduced a hybrid silica-acrylate monolith that could be photopolymerized [58]. Sol-gel reaction of a hybrid monomer, namely 3trimethoxysilylpropylmethacrylate resulted in the localized in-situ synthesis of acrylate-silica monolith. The reversed-phase behavior of such a monolith was confirmed by the separation of two neutral coumarin dyes with efficiency reaching 18 000 plates /meter.

Another approach consisted in the localization of a sol plug, thanks to a push/pull technique using air pressure control. Ishida et al. [59] introduced Chip-LC with a porous monolithic octadecyl-modified silica column prepared within a 15-cm serpentine separation channel of a glass microchip. Pressuredriven LC separation of catechins was carried out and the influence of tapered turns in the serpentine design was evaluated. This LC chip provided plate height of 55 µm.

Another technique consists in the complete filling of the microchip with silica monolith and the subsequent destruction of the monolith in appropriate zones, using NaOH. Silica monoliths from C1-TMOS and C2-TMOS have been synthesized in-situ in glass chips using a 60 °C thermal treatment [60]. The monolith in injection and elution channel was removed by flushing with 3M NaOH. C1-TMOS stationary phase was tuned with 10 % C2-TMOS monomer to increase hydrophobicity and therefore resolution in the separation of nitroaromatic and nitramine explosives. In regards to the low hydrophobicity of this stationary phase, it appears that the incorporation of longer alkyl chains is compulsory in the development of this chromatographic support.

Monoliths have demonstrated many interesting features for the miniaturisation of chromatographic stationary phase. A better understanding of the various synthesis parameters and their relationship with chromatographic behavior is still required for the widespread of these supports in microfluidic environment. But given the ease of fabrication in microchannels, no doubt monoliths will play an important role in the future development of chromatography on chip.

New materials for chromatography

To fabricate size-controlled nanostructure, some approaches have taken advantages of the recent advances in nanotechnologies. The pre-cited COMOSS stationary phases are one example. Another example, as continuous bed, is the use of membranes integrated in microfluidic devices.

Porous anodic alumina was investigated as a stationary phase [61]. Demonstration of chromatographic behavior was achieved on porous alumina membrane incorporated in a chip. The honeycomb array of 300 µm thick with channels of 100 nm diameter was tested and revealed a normal-phase character. Another interesting strategy based on a widely used technique in catalyst fabrication is the idea of "sacrificial" material. In order to design nanopores, Chen's group [62,63] introduced sacrificial silica colloids in microchannels. Silica colloids are grown inside the microfluidic device using evaporation induced self-assembly process. Then the voids between the ordered nanoparticles are filled with SU8photoresist. After curing, the embedded nanoparticles are dissolved using a buffer oxide etch solution.

The resulting continuous bed consists in a bimodal porous structure, with large nanopores corresponding to the removed nanoparticles (300 nm) and smaller interconnecting pores (30 nm) due to the contact between nanoparticles, as shown in figure 9. This nanoporous bed was only used as a sieving matrix for electrophoretic separation of DNA molecules till now. However, due to the efficient and simple control of pore size, this "sacrificial nanoparticle" approach is worth mentioning and would be of interest for chromatographic purposes.

When investigating the various stationary phases that have been implemented in chips, it appears that commercially available designs are aiming towards existing solutions, namely porous silica beads that have given full satisfaction to HPLC users, while the recent years have seen the emergence of a new class of stationary phase, particularly pointing towards smaller dimensions (nanotubes, nanoparticles).

If the realization of particle packed-bed is now fulfilled by few companies, scaling down benchtop instruments, the new materials coming up may invite analysts to apprehend bottom-up way of thinking, especially in terms of fluid manipulation.

III. Instrumental advances and technological issues 1. Benchtop Chip LC: User-friendly NanoLC

NanoLC, which consists in pressure-driven liquid chromatography in capillary columns (10-100 µm i.d.), has well established itself in laboratories as not only the miniaturization of conventional HPLC systems, but more as a complementary technique. The use of capillary format allows the injection of smaller sample quantities. Moreover, the flow rates of few hundreds of nanolitre/min facilitate easy coupling to highly sensitive MS detection. All these features are highly suitable for proteomic area and in other specific domains of application such as enantiomeric analysis, as illustrated by a recent review [64]. Moreover, downscaling of HPLC is also addressing the issue of solvent consumption and waste management which find echoes respectively in economics (as illustrated by the recent acetonitrile shortage) and in the regulation of more environmental-friendly analysis.

There are now on the market as many nanoLC systems as HPLC suppliers. However, it appears that the packed capillary columns with numerous connecting interfaces with injectors and detection systems may impair the robustness of the technique. Moreover, practical considerations need to be Technologies has taken a top-down approach by miniaturizing both injection loop and separation column, limiting the risk of micro leaks and providing a robust instrument for nanoLC [65]. They later demonstrated that dispersions both before and after the chromatographic bed could be reduced when using chip format, illustrating the fact that not only there is a gain in instrumentation facility, but also an improvement in separation performances [30].

The pumping system is an essential instrumental part of Chip-LC. Usually, it consists in conventional pumps that are connected to the chromatographic chip through a split connection [30,48] which use annihilates the argument of on-chip low solvent consumption, as most of solvent volume is deviated to waste to achieve an adequate flow rate. To follow the logical pathway to miniaturization, micropumps able to deliver nanolitre/min rates should be promoted. The fabrication of such micropumps has been a technological challenge. Two interesting reviews on the various options available is cited as references [START_REF] Yun | Micropumps for MEMS/NEMS and Microfluidic Systems[END_REF] and [START_REF] Amirouche | [END_REF]. A first commercial approach in the coupling of micropump and Chip-LC is provided by If gradient elution is nearly systematic in conventional benchtop LC analysis for complex sample, its realization on microchip is not straightforward. As mentioned before, the beginning or the end of the gradient profile requires pumps delivering few nanolitre/min. The gradient delay is another important matter when aiming at high throughput analyses. Even if mixing chamber and tubing are downsized to few nL, the low flow rate increases delay duration. Brennen et al. [68] have addressed this problem by integrating the gradient mixing chamber on chip. The approach realized on Agilent polyimide chip consists in the microfabrication of so-called "timing channels", which are parallel channels of various lengths, interfaced through a single point to the LC column, as shown in Figure 10. At initial time, the channels are filled with starting mobile phase A. As the analysis is carried out, more eluent mobile phase B enters every channel. Every time a channel is filled with B, the overall mobile phase composition sees its B-content increases. The resulting step profile can be assimilated to a linear profile if the increments in length between channels are small enough. The actual Agilent gradient rate is induced by a single pump controlling B solvent entry in the parallel channels and governed by microfabricated restrictors. Unfortunately, three other pumps are required to condition "timing channels", flush LC chip and inject sample, which adds complexity to the instrumental setup. Through this approach, the delay duration is minimized (delay volume of few nanolitre) by directly connecting gradient chip with LC chip, while the gradient volume (volume used to change from 100 % A to 100 % B) is permanently defined by the microchip design.

Chip-LC is a mature technique which has recently seen the emergence of commercially available instruments dedicated to its miniaturized environment. Miniaturization on chip has brought solutions in the downsizing of dead volumes, either before (on-chip injection, on-chip gradient) or after the column (direct coupling to ESI-MS). The resulting performances are expected to be remarkable, proving Chip-LC to be the technique/instrument of choice in the analysis of complex sample such as proteomics.

Portable Chip-LC: a higher level of integration

The development of liquid chromatography on chip can also be seen as an adequate answer to on-site analysis. Portable analytical systems can be required for analysis directly conducted at the point of sampling to provide a fast response to a very specific problem. Various situations can be encountered such as point of care or point of consumption. As point of care is concerned, the market for portable, user friendly analysers has already exhibited interest towards microfluidics, as illustrated by the many commercially available miniaturized assays (pregnancy, diabetes, iron content). Diagnosis or followup of medical treatment will in the future require the realization of a full analytical process (sample treatment, separation, detection) in a non-chemist environment (costumer or trained person). Point of consumption is another area avid of miniaturized on-site analysis, to control food freshness or water quality with a fast response time.

Chip-LC systems are betting on high performances in complex sample analysis. If the user-friendly setup is indisputable through commercial instruments (docking station, automatic chip alignment), the overall analytical chain remains a cumbersome, benchtop-sized system, usually involving two or three external pumps and MS detector. The instrumentation remains impressive and hardly portable.

Miniaturization of pumping systems is still in its infancy and the cost of micro valves prohibits their discarding. Miniaturization of the detection technique is another aspect that we will not address in this discussion. The generation of fluid flow using electrochemistry or electroosmosis is perfectly suitable to miniaturization, as it delivers flow rates in the range of few hundreds nanolitre/min. On the technological point of view, it requires only sets of electrodes and is running off batteries. Involving cheap technology (no moving parts) it is directly integrated on chip and can be discarded after use.

Electrochemical pumps are based on the generation of gas in enclosed reservoirs through electrolysis.

This mechanism requires low voltages, builds up pressures around 15-20 bars and can induced hydrodynamic flow rates in the range of 80 nL/min through an open-tubular column [69] or a packed bed [70]. In the most accomplished reduction of dead volume and delay volume, Xie and colleagues [70] presented an electrochemical pump with two 20 µl -reservoirs linked to a 1.5 nL static mixer to perform a mobile phase gradient through a 1.2 cm long packed bed. The corresponding delay volume was only 15s. In comparison, the gradient time was set to 30min.

Electroosmotic pumps are another option to generate a flow rate under electrical actuation. Through the creation of an electrical double layer on charged surface, the fluid is propelled with a velocity directly related to electrical field. The resulting flow rate is then directed downstream in a field-free region where the chromatographic bed sits. Integrated EOF pumps have been implemented to Chip-LC systems by Lazar et al. [71] and by Collins et al. [72]. In the first case, the EOF pump consists in a series of 200 unpacked nanochannels etched in glass to provide the source of electroosmosis. The chosen configuration allows the implementation of gradient elution, EOF valving and injection in the 2 cm-long column packed with 5µm C18 silica particles. The main concern focused on the backpressure that is issued from the chromatographic bed and requires a consistent hydraulic resistance of the nanochannels.

This concern was partially answered by Collins' group [72] who used a design including two stationary phases, as illustrated in Figure 11. The first bed is packed with silica particles to generate EOF, while the downstream separation bed is filled with silica monolith, well known for its high permeability. This setup minimizes backpressure from the field free region and therefore optimizes the delivered flow rate, 5.30 nL.min -1 . kV -1 in 20 mM Tris, 60 % acetonitrile solution, in this case.

A limitation to the spread of electrokinetic pumps is the low backpressure that they can withstand.

Indeed, if the material generating the electroosmotic flow is too permeable in comparison with the following chromatographic bed, backpressure from the head of the column can result in hydrodynamic backflow into the EOF/ electrochemical pump, reducing the actual flow rate. Investigations will proceed to match pumping systems and downstream columns.

Chip-EC: both portable and disposable

If portability and user-friendly character are essential in the investment of this analysis-on-site area, the fact that health is concerned makes the disposability another reason to miniaturize chromatography.

If electrokinetic pumping-LC illustrate a solution towards total integration, it involves the synthesis of two different beds separated by a specific membrane, with sufficient hydrodynamic restrictions to avoid siphoning between solvent reservoirs or back flush upstream from LC column. Moreover, the use of hydrodynamic flow in injection and separation units makes tightness a crucial point. The overall technology is complex and may be costly.

Electrochromatography technique appears to be the simplest answer to carry out chromatography on the field in disposable devices. Not only technological requirements (electrodes, batteries) are suitable to portability, but the absence of injection valves (as opposed to Chip-LC) by the integration of electrokinetic injection on-chip allows the throwing-away of the overall analytical chain. This ability to discard both injectors and columns i.e. the overall parts that have come into contact with sensitive sample is especially interesting for cross contamination issue (biological samples) or specifically damaging domains, like nuclear environment.

The answer proposed by electrochromatography is to combine both pump and column, by initiating electroosmosis directly from the chromatographic stationary phase. On the instrumental point of view, it is the simplest unit as only one stationary phase is required and proper sealing of reservoirs is less essential than in hydrodynamic flow. Electrochromatography exhibits specific features when compared to pressure-driven chromatography [73]. In electroosmosis, the fluid movement arise from the surface of the material, which theoretically results in identical velocity inside and outside the porous structure.

This results in a minimal eddy diffusion (A-term) and reduces mass transfer contribution to band broadening (C-term) since the volume of stagnant mobile phase is reduced. It may counterbalance any discrepancy in packing homogeneity as well. Moreover, as the flow rate is independent of the particle size, it can be interesting to use smaller and smaller particles to achieve better chromatographic performances, which has dramatic consequences in pressure-driven technique. Sub-1 µm particles were actually investigated in CEC [74] and many new stationary phases within the sub-micron size range that are now developed in microchannels will find optimal working conditions in electrochromatography mode [4,75,76].

Another point in favour of electrochromatography is the mixed-mode separation mechanism that occurs during analysis. This argument becomes sterile if one considers that the two techniques are complementary and that the disposable character of Chip-EC means that the device will not be subject to the same high throughput as laboratory-based Chip-LC.

Another challenge in electrochromatography is the dependency of flow velocity to mobile phase composition. This results in a real challenge to program gradient elution profile if complex samples are considered. Ramsey's group focus on this problem since the late 90's [5,78]. Using an open-tubular format as the separation column, they designed a solvent programming where the separation electrical field remains constant over analysis duration, while the amounts of solvent A (less eluent) and B (more eluent) varies as a function of time. Three elution schemes are evaluated: isocratic elution, step gradient and linear gradient. If the first results were promising, the linear velocity developed through the analysis duration evolves as the mobile phase composition changes.

Recently, Watson et al. [49] address this problem by using velocity-matched pairs of buffers. In the design illustrated as Figure 12, an acrylate monolith is cast in the separation region to provide the stationary phase, while two inlets are connected to a serpentine mixer to achieve proper solvent composition. The authors have chosen to tune ionic strength in buffers in order to maintain constant flow mobility when varying acetonitrile content. This was done by calibration of the individual velocities to properly select pairs of velocity-matched buffers. When the two inlet voltages were ramped according to elution program (fig 12b), the overall flow rate remained constant, as both electrical field and electroosmotic mobilities were fixed in the separation channel.

If Chip-EC has not yet achieved robustness of benchtop Chip-LC, it addresses issues of portability and disposability and proved to be a simple solution to simple problems. Currently limited to isocratic mode, this technique has recently seen elegant advances to establish gradient elution at constant mobile phase velocity. Despite exhibiting a high potential for the future of separation science, especially towards novel stationary phases and sub-microns particle size, its widespread has been so far hold back, probably due to the lack of commercially available Chip-EC system. repulsed solutes localized at the centre acquire a higher velocity than less charged solutes localized near the channel walls. This velocity was proved to depend on Debye Length, i.e. ionic strength. In the light of the above considerations, femto liquid chromatography can be considered as an extreme example of Chip-LC, involving no stationary phase but the interaction (repulsion) between solutes and walls. This example may not reach everyday user, but it highlights the aptitude of miniaturization to initiate novel separation mechanisms.

Alternative choices

IV. Conclusion

Outstanding achievements have been accomplished in the past five years, both in the development of novel stationary phases and in the design of innovative analytical microsystems. This modern field of research that was liquid chromatography on chip has taken a step further towards maturity, leaving the experimental stage to gain the market place. If pressure-driven Chip-LC has found its place in laboratories as a well-established miniaturized version of HPLC, Chip-EC is still facing challenges and

has not yet fully expressed its potential towards simplicity and portability.

Further Chip-LC investigations will apprehend complex analytical problems and will carry on with the implementation of existing HPLC features (2D, high temperature, high pressure) to miniaturized environment. On the other hand, electrochromatography has never breakthrough in capillary format [73], but is easily implemented in chip-format, which augurs a very promising future. Chip-EC has not yet found its "killer application" and besides its perfect suitability towards nanoworld (nanoparticles, nanochannels), is still undervalued.

F

I

  . Introduction ...................................................................................................................................... 2 II. A brief overview of stationary phases in chips ................................................................................ 41. Open-channel chromatography ................................................................................................ 4 2. COMOSS, pillars and nanotubes.............................................................................................. 6 3. Packed Particles........................................................................................................................ 8 4. Monoliths and continuous structures........................................................................................ 9 5. New materials for chromatography........................................................................................ 12 III. Instrumental advances and technological issues ........................................................................ 13 1. Benchtop Chip LC: User-friendly NanoLC ........................................................................... 13 2. Portable Chip-LC: a higher level of integration..................................................................... 15 3. Chip-EC: both portable and disposable.................................................................................. 17 4. Alternative choices ................................................................................................................. 18 IV. Conclusion.................................................................................................................................. 19 V. References ...................................................................................................................................... 20

  developed an array of native COC pillars (here, TOPAS ® ) of 4 µm wide by 8 µm long by 5 µm deep, generating separation channels of 2 µm wide for electrochromatography purposes (Fig 3). The hydrophobic character of polymeric substrate has been cleverly exploited to use the microdevice substrate as a stationary phase. Not only native TOPAS ®

  to this novel stationary phase, with modification of the selectivity when separating three dsDNA fragments by electrochromatography. Interestingly, the plug of solutes was less subject to band broadening in the carbon nanotubes stationary phase than in open channel. It was suggested by the authors that the very laminar flow field generated in the nanotubes environment was less susceptible to perturbation for dispersion. Efficiency in such a novel stationary phase reached up to 3.8 x 10 5 plates/meter.

  packing. This packing procedure allowed immobilization of 3 µm particles in a reproducible way in a taper 16 µm wide at the narrowest end. If efficiencies reached 290 000 plates/m

  is present in the porogen solution. This phenomenon may be due to the highly hydrophobic character of COC which disturbs homogeneity of the polymerization mixture. On the opposite, polymerization occurs if the porogen is constituted of only organic solvents (usually alcohols).

Formatted:

  Bullets and NumberingFormatted: English (U.K.)

  a user-friendly nanoLC system, such as micro leaks and band broadening due to external effects. An argument towards integration on chip is the possibility to achieve zero deadvolume connections. With the first commercially available integrated LC-on-chip solution, Agilent

  Eksigent company[34], whose cHiPLC Nanoflex system presents a leak-less connecting station between a splitless micropump and a glass chip for Chip-LC purposes. The Microfluidic Flow control consists in two pneumatic pumps used to amplify pressure in solvent reservoirs. Two flow sensors measure the flow coming out of each individual pump heads and use the feedback to control the driving pressure. Performance of such a pump relies on fast response of the flow sensor and proper feedback from the control loop. The risk underlies when very low flow rate is requested from one pump at the boundaries of the gradient profile.

  made of 20 channels to get a smooth profile, imposing a 2.4 µl gradient volume. Flow

  to on-site chromatography consists in the use of electrically activated pumps.

  Figure 1

Figure 2 Wiley

 2 Figure 2

Figure 4 Wiley

 4 Figure 4

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 10

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 4. Van Deemter curves of C440 (unretained compound -circle) and C480 (retained compound -square) in mobile phase compositions of 50/50 and 30/70 (v/v) methanol/water,respectively, obtained by pressure-driven chromatography on unmodified COC pillars[22]

Figure 7 .Figure 8 .Figure 9 .

 789 Figure 7. SEM image of vertically aligned carbon nanotubes directly grown in microchannel.Chemical structure of 1-pyrenedodecanoic acid modified carbon nanotube[26]

Figure 10 . 68 ]Figure 11 .Figure 12 .

 10681112 Figure 10. Left -Schematic of gradient generation concept with 1-inlet, 2-restrictors, 3timing channels, 4-outlet. Right -photograph of a 20-stage Agilent gradient generation chip. [68]

  By combining electrophoresis with a stationary phase interaction, this technique is able to separate both neutral and charged compounds. Besides this, He et al.[77] showed that CEC can improve LC and/or CE separation by a proper selectivity on proteins, such as ovalbumin.Of course, when Chip-EC and CEC have not break through as competing separation techniques on the LC market, it is because drawbacks are still multiple. While hydrodynamic flow rate is externally monitored, electroosmotic flow rate is directly dependent on surface chemistry, mobile phase and electrical field in the separation channel. Electrical field finds limitations in Joule heating and instrumental resistance (usually few kV/cm), while surface chemistry can be altered over time by pH change, solute adsorption, making this technique less robust than pressure-driven chromatography.

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  It has to be mentioned some non-conventional driving forces that have been investigated to achieve liquid chromatography on chip. If these methods may appear as epiphenomena, they are fully taking advantages of the miniaturized environment of chips to achieve chromatography and therefore should be considered as innovative.Shear-driven chromatography is based on the displacement of a channel wall in respect to a nonmoving coated wall. If high efficiencies and fast analysis have been achieved using this technique, research in this area is mostly restricted toBaron and Desmet group due to technical difficulties working in nanochannels [79]. Evaporation driven flow vas also explored as a new simpler way to move mobile phase through the chromatographic bed [80]. However, this mode was subjected to a number of external parameters and full control of the flow rate was tedious, involving a permanent air stream over the evaporation zone. Aiming at ultra miniaturization is the very specific work by Kitamori et al. [81] who described a chromatographic separation realized in the extended nanospace. The size of the quartz nanochannels (270 * 280 nm²) attains the range of Debye length, which results in a repulsion of charged solutes towards the centre of the nanochannel. The pressure-driven flow leading to a parabolic profile,

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  Luedtke, S., Adam, T., von Doehren, N., Unger, K.K., J.Chrom A, 2000, 887, 339-346 [75] de Mello, A., Lab Chip, 2002, 2, 48N -54N [76] Stachowiak, T.B., Svec, F., Fréchet, J.M.J., J. Chrom A, 2004, 1044, 97-111 [77] He, B., Ji, J., Regnier, F.E., J. Chrom A, 1999, 853, 257-262 [78] Kutter, J.P., Jacobson, S.C., Matsubara, N., Ramsey, J.M., Anal. Chem., 1998, 70, 3291-3297 [79] Fekete, V., Clicq, D., De Malsche, W., Gardeniers, H., Desmet, G., J. Chrom A, 2007, 1149, 2-11 [80] Goedecke, N., Eijkel, J., Manz, A., Lab Chip, 2002, 2, 219 -223

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  ). Reservoir voltages on the low-ACN (2%) (red, solid) and high-ACN (60 %) (blue, dashed) buffer inlets were varied as indicated on gradient programming scheme.[49]

	Liquid Chromatography on chip Liquid Chromatography on chip
	Karine Faure Karine Faure
	Laboratoire des Sciences Analytiques, Laboratoire des Sciences Analytiques, UMR UCBL -CNRS 5180, Université de Lyon UMR UCBL -CNRS 5180, Université de Lyon Université Claude Bernard Lyon 1 Université Claude Bernard Lyon 1 43 boulevard du 11 Novembre 1918 43 boulevard du 11 Novembre 1918 69622 Villeurbanne, France 69622 Villeurbanne, France karine.faure@univ-lyon1.fr karine.faure@univ-lyon1.fr
	Abbreviation list
	F o r r o F AMPS, 2-Acrylamido-2-methylpropane sulfonic acid; CD, cyclodextrin;
	P P
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	e r e r
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Chip

-LC, pressure-driven liquid chromatography on chip; Chip-EC, liquid electrochromatograhy on chip; COC, cyclic olefin copolymer; COMOSS, collocated monolith support structure; Dns-AAs, dansylated amino acids; EDMA, ethylene dimethacrylate; EOF, electroosmotic flow; ESI, electrospray ionization; PDMS, polydimethylsiloxane; TEOS, tetraethyl orthosilicate.

Wiley-VCH ELECTROPHORESIS

20

V. References

Acknowledgments :

The author wishes to thank Gerard Cretier for helpful discussion and proof reading The author acknowledges financial support by the Research National Agency (ANR), project ANR-07-PCVI-0036-01 (''D-aminoChip'').


Audrey Guilmatre

Solenn Legallic

Gary Steel

Alecia Willis

Gabriella Di Rosa

Alice Goldenberg

Valérie Drouin-Garraud

Agnès Guet

Cyril Mignot

Vincent Des Portes

Gabriella Di

Vincent Des

Vassili Valayannopoulos

Lionel Van Maldergem

Jodi D Hoffman

Claudia Izzi

Caroline Espil-Taris

Simona Orcesi

Luisa Bonafé

Eric Le Galloudec

Hélène Maurey

Christine Ioos

Alexandra Afenjar

Patricia Blanchet

Bernard Echenne

Agathe Roubertie

Thierry Frebourg

David Valle

Dominique Campion
email: dominique.campion@univ-rouen.fr

John Wiley

Type I hyperprolinemia: genotype/phenotype correlations

Keywords: PRODH, Type I hyperprolinemia, 22q11, POX enzymatic activity PRODH, type I hyperprolinemia, 22q11, POX enzymatic activity

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

of PRODH variations described in HPI patients, second to assess the functional effect of PRODH mutations on POX activity and finally to establish genotype/enzymatic activity correlations in a new series of HPI patients. 8/14 variants occurred at polymorphic frequency in 114 controls. POX activity was determined for 6 novel mutations and 2 haplotypes. The c.1331G>A, p.G444D allele has a drastic effect whereas the c.23C>T, p.P8L allele and the c.[56C>A; 172G>A], p.[Q19P; A58T] haplotype result in a moderate decrease in activity.

Among the 19 HPI patients, 10 had a predicted residual activity <50%. 8 out of 9 subjects with a predicted residual activity ≥50% bore at least one c.824C>A, p.T275N allele, which has no detrimental effect on activity but whose frequency in controls is only 3%. Our results suggest that PRODH mutations lead to a decreased POX activity or affect other biological parameters causing hyperprolinemia.

Introduction

Type I hyperprolinemia (MIM# 239500) is characterized by an increase in plasma proline level in absence of urinary pyrroline-5-carboxylate (P5C) (typical of type II hyperprolinemia).

It was initially described in 1962 as a nephropathy associated with cerebral dysfunction [START_REF] Efron | Familial hyperprolinemia. report of a second case, associated with congenital renal malformations, hereditary hematuria and mild mental retardation, with demonstration of an enzyme defect[END_REF][START_REF] Schafer | Familial hyperprolinemia, cerebral dysfunction and renal anomalies occuring in a family with hereditary nephropathy and deafness[END_REF]. The association with renal disease was fortuitous and was due to a recruitment bias since patients with nephropathy were preferentially screened by amino acid chromatography at this time. As a result of the analysis of new families with HPI ten years later, it was considered that hyperprolinemia was a benign condition without mental retardation or renal dysfunction [START_REF] Mollica | Hyperprolinaemia: a disease which does not need treatment?[END_REF]. The gene associated with this condition, named PRODH (MIM# 606810), was mapped in 1997 on 22q11 [START_REF] Campbell | A human homologue of the drosophila melanogaster sluggish-a (proline oxidase) gene maps to 22q11.2, and is a candidate gene for type-i hyperprolinaemia[END_REF] and encodes for proline oxidase (POX), the first enzyme involved in proline catabolism. Its expression is limited to liver, brain and kidney. In 1999, a mouse model deficient for POX was described. These mice present behavioral problems, especially a sensory motor gating impairment, which is an endophenotype of schizophrenia [START_REF] Gogos | The gene encoding proline dehydrogenase modulates sensorimotor gating in mice[END_REF]. A few years later, [START_REF] Humbertclaude | Is hyperprolinemia type i actually a benign trait? report of a case with severe neurologic involvement and vigabatrin intolerance[END_REF]]described a case with severe neurologic impairment and raised the question whether HPI really was a benign trait in humans, which opened up again the discussion.

In 2003, we described [START_REF] Jacquet | The severe form of type i hyperprolinaemia results from homozygous inactivation of the prodh gene[END_REF] a recurrent 350kb deletion encompassing the PRODH and DGCR6 (MIM# 601279) genes, mediated by low copy repeats surrounding this region, which was subsequently found to be present in 1/250 individuals in the general population [START_REF] Guilmatre | Recurrent rearrangements in synaptic and neurodevelopmental genes and shared biologic pathways in schizophrenia, autism, and mental retardation[END_REF][START_REF] Ohtsuki | Failure to find association between prodh deletion and schizophrenia[END_REF]. Numerous missense mutations in the PRODH gene have also been described, most of them appearing recurrently because of a gene conversion between PRODH and a non-processed pseudogene located 1.4 Mb telomeric on chromosome 22 [START_REF] Liu | Genetic variation in the 22q11 locus and susceptibility to schizophrenia[END_REF]. A functional characterization of POX enzymatic activity 5 was established demonstrating that some of these mutations had a severe impact (> 70% reduction) on its activity whereas others had a mild or moderate effect [START_REF] Bender | Functional consequences of prodh missense mutations[END_REF].

Further genetic studies in 3 patients with mental retardation, epilepsy and very high plasma proline level allowed us to establish that the severe form of HPI was a recessive disorder resulting from homozygous PRODH/DGCR6 deletion or mutations with a drastic effect on enzymatic activity [START_REF] Jacquet | The severe form of type i hyperprolinaemia results from homozygous inactivation of the prodh gene[END_REF]. In accordance with the mouse model phenotype, hyperprolinemia was then shown to be a risk factor for schizoaffective disorder [START_REF] Jacquet | Hyperprolinemia is a risk factor for schizoaffective disorder[END_REF]. The link between hyperprolinemia and cognitive or psychiatric troubles was further strengthened by the study of patients with velo cardio facial syndrome (VCFS) that results from a 1.5-3Mb deletion on chromosome 22q11. Although all these patients are hemizygous for the PRODH gene, only 30-50% have elevated plasma proline level [START_REF] Goodman | Hyperprolinaemia in patients with deletion (22)(q11.2) syndrome[END_REF][START_REF] Raux | Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome[END_REF], while 50% have mental retardation and 30% psychosis [Murphy et al., 1999]. We have shown that in VCFS increased plasma proline level (in association with the COMT (MIM# 116790) genotype) was a risk factor for psychosis and that prolinemia was inversely correlated to IQ [START_REF] Raux | Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome[END_REF]. Based on the functional predictions, this study also showed that a reduction of at least 50% of the enzymatic activity was required to produce mild to moderate hyperprolinemia while severe hyperprolinemia was generally associated with a near complete loss of enzymatic activity. In addition to these observations, PRODH/DGCR6 deletion and mild hyperprolinemia were recently found at a high frequency in autistic subjects [START_REF] Guilmatre | Recurrent rearrangements in synaptic and neurodevelopmental genes and shared biologic pathways in schizophrenia, autism, and mental retardation[END_REF].

Despite this large body of data, several points remain to be clarified. In particular, correlations between PRODH genotype and various levels of hyperprolinemia, ranging from mild to severe, remain poorly understood. In the present report, three issues were examined: first we measured in controls the allelic frequency of PRODH variations found in HPI patients, second we assessed the functional effect of previously uncharacterized mutations or haplotypes on

Material and methods

This study was approved by the Ethics Committee of Rouen University Hospital.

Controls

To establish the allelic frequency of PRODH (NM_016335) variants, we genotyped 114 healthy caucasian subjects originating from France (46 men, 68 females) previously included in a study aimed to establish the normal values of plasma proline in the adult population [START_REF] Jacquet | Hyperprolinemia is a risk factor for schizoaffective disorder[END_REF]. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

Patients with HPI

Blood samples from patients with HPI were sent to our laboratory from centers expert in the diagnosis of Autism or mental retardation in order to analyze the PRODH gene. All patients were examined by experienced clinicians and psychiatric diagnoses were made according to DSM IV criteria. Biological investigations included MRI imaging, karyotype, Fragile X testing and amino-acid chromatography. Plasma proline levels were determined by ion exchange chromatography after overnight fasting. After informed consent was obtained, DNA was extracted from peripheral lymphocytes. Patients as well as their parents were genotyped in order to obtain the phase of the mutations.

PRODH sequencing and QMPSF

PRODH exons were sequenced using PCR conditions and primers previously described [START_REF] Jacquet | Hyperprolinemia is a risk factor for schizoaffective disorder[END_REF][START_REF] Raux | Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome[END_REF]. In some subjects in which PRODH exon 7 could not 7 be amplified , due to indels in both flanking introns, the presence of the c.824C>A, p.T275N variation was assessed using the forward primer 5'TGCAGTTCTCAGAGGTGCTG 3' in combination with the previously described reverse primer.

PRODH deletions were characterized by quantitative multiplex analysis of short fluorescent fragments (QMPSF), as previously described [START_REF] Jacquet | Prodh mutations and hyperprolinemia in a subset of schizophrenic patients[END_REF].

Array CGH

DNA samples were analyzed using Human High-Resolution Discovery Microarray Kit 4x180K (Agilent Technologies, Santa Clara, California, USA), using standard recommended protocols. Non commercial genomic DNA pool of 10 control individuals was used as reference. Hybridization results were extracted with Feature extraction software and analyzed using Agilent's DNA-analytics software. The data were processed using ADM-2 algorithm, with the threshold set at 6.0 SD.

Functional assay for POX activity

We purchased restriction enzymes and buffers from New England Biolabs: Pfu-Turbo DNA polymerase from Stratagene and chemicals from Sigma. 14 C-proline was obtained from New England Nuclear and purified prior to use by ion-exchange chromatography on Dowex AG 50w-8x hydrogen form (BioRad) [START_REF] Phang | Disorders of proline and hydroxyproline metabolism[END_REF]. To assay green fluorescent protein (GFP) fluorescence, we used a Zeiss LSM 510 Meta confocal laser-scanning microscope.

Functional test was realized as previously described [START_REF] Bender | Functional consequences of prodh missense mutations[END_REF]. Briefly, the PRODH ORF divided into three fragments previously cloned in the pBluescript KS vector 8 fragment into the pTracer expression construct and resequenced the construct to verify the presence of the mutation and the integrity of the ligations. For all expression studies, we utilized a subclone (CHO-K1-C9) of CHO-K1 cells that lack endogenous POX activity [START_REF] Valle | Proline inhibition of pyrroline-5-carboxylate reductase: differences in enzymes obtained from animal and tissue culture sources[END_REF]. For electroporation, we used 30 µg of the indicated plasmid DNA and 350 volts/ 400 Ω/960 µF in 300 µl of growth medium containing 1.25% DMSO and 5-7 × 10 6 cells.

After 48 h, we harvested the cells by washing the monolayer with PBS and scraping them into cold PBS. The cells were collected by centrifugation at 480 x g and were resuspended in 0.1 M KPO 4 (pH 7.2). The cells and their organelles were disrupted by sonication for 1 min at a setting of 25% (Branson Sonifier 450 [Branson Ultrasonics]). Total protein was determined with the Pierce BCA protein assay (Pierce). POX-specific activity (nmol/prod/mg/hr) was assayed radioisotopically, as described elsewhere [START_REF] Phang | A radioisotopic assay for proline oxidase activity[END_REF]. To account for variation in transfection efficiency, we expressed the data as a percentage of wild type (specific activity of mutant allele/transfection efficiency)/( specific activity of wild-type allele/transfection efficiency). For alleles with severe reduction in POX activity, we repeated the assay with the addition of 1 mM FAD. All samples were assayed in triplicate in independent transient transfection experiments.

Ex vivo splicing assay using a splicing reporter minigene

This assay was used to test the effect of the p.T275N variant in PRODH exon 7 splicing. This assay was realized in both Hela and ShSY5Y cells using the same protocol [START_REF] Tournier | A large fraction of unclassified variants of the mismatch repair genes mlh1 and msh2 is associated with splicing defects[END_REF], with DMRIE-C (Invitrogen) as the transfection reagent for the SH-SY5Y cells. Primer sequences are available upon request. The genotyping of 114 controls without psychiatric or neurological disorders (four of them having plasma proline level above the upper threshold, range 389-482 µmol/l) allowed us to determine in a control population the frequency of missense mutations that were present in HPI patients [START_REF] Raux | Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome[END_REF] (and this study) (Table 1). For each mutation, the distribution of genotypes followed the Hardy-Weinberg equilibrium. Among the 14 variations we analyzed, 8 occurred at a polymorphic frequency (> 0.01), two of them (c.56C>A, p.Q19P and c.553T>C, p.R185W) being very common with a minor allele frequency of 0.48 and 0.36, respectively.

Results

Frequency of PRODH variations in controls

Functional consequences of PRODH missense mutations

In addition to data already reported, that are summarized in Supp Table S1 [START_REF] Bender | Functional consequences of prodh missense mutations[END_REF], we describe here the results of functional analyses for a new series of PRODH missense mutations (c.23C>T, p.P8L ; c.32G>C, p.R11P ; c.88C>T, p.P30S ; c.172G>A, p.A58T ; p.T275N and c.1331G>A, p.G444D) and haplotypes (c.[56C>A; 172G>A], p.[Q19P; A58T] and c.[56C>A; 88C>T], p.[Q19P; P30S]) (Figure 1). We show that the p.G444D substitution has the most drastic effect resulting in only 27% of residual enzymatic activity. p.P8L and the haplotype p.[Q19P; A58T] have both moderate effect. However the other mutations have no effect on enzymatic activity or even a tendency to increase it. As previously described for other haplotypes [START_REF] Bender | Functional consequences of prodh missense mutations[END_REF] the POX activity encoded by the p.[Q19P; A58T] allele was dictated by the most severe missense mutation with no evidence of intra allelic complementation. By contrast, analysis of the p.[Q19P; P30S] haplotype showed that the p.P30S variation fully complemented the deleterious effect of the p.Q19P mutation. 2a. Most of these patients present with psychiatric and cognitive troubles and epilepsy. MRI findings were normal except for two patients who displayed agenesis of the corpus callosum, symmetrical temporal lobe hypoplasia, thalamus fusion and right opercular dysplasia, respectively. Dysmorphism was noted in 4 patients who were all negatively screened for DNA rearrangement by aCGH, as was patient 109 who had an abnormal MRI but without dysmorphism.

In this series of patients, abnormal plasma proline levels ranged from 1667 to 327 µmol/l with, for several subjects, considerable variation between two measures obtained at different time (Table 2b). Based on the results of functional assay, four subjects had a genotype predicted to result in a drastic loss of enzymatic activity. In accordance with these predictions, they all present with severe hyperprolinemia. Overall, only 10/19 patients had a predicted residual activity <50% which, according to our working hypothesis [START_REF] Raux | Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome[END_REF], is required to produce hyperprolinemia. Strikingly, 8 out of 9 subjects with a predicted residual enzymatic activity ≥50% bore at least one p.T275N allele, which has no effect on enzymatic activity but whose frequency in the control population is only 3%. Using an ex vivo splicing assay to further analyse the effect of this variant, we observed no effect on the splicing pattern of PRODH exon 7 (data not shown).

Discussion

All patients included in this report present cognitive impairment and/or behavioral problems.

Epilepsy was also frequent. It should however be stressed that this study was not designed to 11 formally assess the clinical spectrum of HPI. Thus, although consistent with the view that HPI is associated with psychiatric and neurologic disabilities, our data may just reflect a recruitment bias since subjects presenting such conditions are more likely to be screened for inborn error of amino acid metabolism than normal subjects.

Nonspecific dysmorphic features were present in a subset of patients raising the question of a putative link between HPI and syndromic MR. In these patients, array CGH analysis failed to document the co-occurrence of another genetic abnormality that can explain these physical features. Thus, it remains unclear whether dysmorphism may be a component of the HPI syndrome or whether another as yet undetected genetic defect is present in this subset of patients.

Concerning the relationship between plasma proline and the predicted POX activity, a rather poor correlation was observed. However, as previously noted [START_REF] Bender | Functional consequences of prodh missense mutations[END_REF] our functional assay circumvent any effect these mutations might have on RNA splicing or protein stability. It is thus conceivable that several variations may be wrongly classified as benign on the basis of this sole test. In a previous report concerning VCFS patients we had already noted the unexplained enrichment of the c.1292G>A, p.R431H mutation in patients with high proline levels [START_REF] Raux | Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome[END_REF]. In the present report, the increased frequency of the functionally neutral p.T275N mutation in HPI patients is noteworthy, pointing out that it is indeed a probable pathologic variant. Since this variation does not modify the splicing pattern of exon 7, its biological effect remains elusive and it is most likely in linkage disequilibrium with another functional variant. Indeed, DNA variations in the regulatory regions or in introns of PRODH may influence transcription or splicing of the gene. A polymorphism located in the 5' region of PRODH and resulting in a 1.5-fold increase in promoter activity has been described [START_REF] Hoogendoorn | Functional analysis of polymorphisms in the promoter regions of genes on 22q11[END_REF]. However, genotyping this polymorphism in our As previously noted, plasma proline is subject to fluctuations over time in most patients, thus requiring, as a rule, repeated determinations. However, several patients bearing a potentially deleterious genotype had only mild hyperprolinemia on repeated measures. Since it seems unlikely that such a slight increase in proline level is sufficient to produce a detrimental effect, this result questions the value of plasma proline level determination as an estimate of CNS proline value. Because peripheral proline cannot gain access to the central nervous system (CNS) across the blood-brain barrier [START_REF] Hawkins | Structure of the blood-brain barrier and its role in the transport of amino acids[END_REF], blood and brain proline form two largely independent pools. Brain proline, the concentration of which in the cerebro spinal fluid (CSF) is low (4.2 µmol/l), is not subject to nutritional fluctuations. Its level depends on local synthesis from ornithine and glutamate and from degradation of collagen of the extra cellular matrix. In severe HPI [START_REF] Efron | Familial hyperprolinemia. report of a second case, associated with congenital renal malformations, hereditary hematuria and mild mental retardation, with demonstration of an enzyme defect[END_REF][START_REF] Humbertclaude | Is hyperprolinemia type i actually a benign trait? report of a case with severe neurologic involvement and vigabatrin intolerance[END_REF] as well as in PRODH -/-mice [START_REF] Gogos | The gene encoding proline dehydrogenase modulates sensorimotor gating in mice[END_REF], CNS concentration of proline is elevated.

Unfortunately, CSF data are not available for our patients.

Another point to be kept in mind when interpreting slight increases in plasma proline is that normative values of plasma proline are lower in children as compared with adults.

Accordingly, in mice PRODH transcripts are expressed at higher level in the young brain as compared with the mature brain [START_REF] Maynard | A comprehensive analysis of 22q11 gene expression in the developing and adult brain[END_REF]. Physiologically, the fine tuning of PRODH expression facilitates proline catabolism during periods in which most refinement and stabilization of synaptic connections occur. Therefore, it can be hypothesized that any genetic defect causing even a modest increase in proline levels during critical periods of development may have a dramatic effect on CNS functioning.

  and third we attempted to establish correlations between genotype and enzymatic activity in a new series of HPI patients.

(

  Stratagene) was used to perform the mutagenesis and the wild-type PRODH cDNA previously cloned into pTracer (Invitrogen) was used for expression analysis. We mutagenized the ClaI/BstEII-modified PRODH cDNA in pBluescript KS by PCR using the QuikChange Mutagenesis Kit (Stratagene). Dependent on the position of the desired mutation, we subcloned either the mutagenized KpnI/ClaI or mutagenized ClaI/BstEII

  correlations in patients with type I hyperprolinemiaClinical and demographic characteristics of a novel series of HPI patients whose PRODH genotype and plasma proline level have been determined are displayed in Table

  reveal any relationship between the low transcriptional activity allele and HPI (data not shown). Characterization of further variations in these regulatory regions is thus required.

  Figure and table legends

Table 1 :

 1 Frequency of missense mutations in controls PRODH (NM_016335) Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1. Table 2: Phenotypic and genotypic features of patients with Type I hyperprolinemia a: clinical features F: female; M: male; age (years); #: born from consanguineous parents; * corpus callosum agenesis, symmetrical temporal lobe hypoplasia, thalamus fusion; ** right opercular dysplasia MRI: Magnetic Resonance Imaging; aCGH: array Comparative Genomic Hybridization AF: autistic features; AP: atypical psychosis; At: ataxia; Au: autism; D: dyslexia; E: epilepsy; LD: learning disorder; MR: mental retardation; PE: pharmacoresistant epilepsy; PMD: Psychomotor delay. + hypertelorism, telecanthus, high palate, ligamentar hyperlaxity; ++ frontal bossing, thin lips, syndactyly 2 rd -3 rd toes; +++ high forehead, frontal bossing, micrognathism, thin upper lip, short nose, short stature , microcephaly -2.5 SD; ++++ short nose with anteverted nostrils, prominent ridging of the metopic suture, long and flat philtrum and thin upper lip, small chin, hypertelorism, strabismus. b: molecular features PRODH (NM_016335); WT: wild type; del: deletion Fasting abnormal plasma proline values: age >18 years: males > 377 µmol/l, females > 316 µmol/l; 5 years>age <18 years: > 270 µmol/l; age < 5 years: > 235 µmol/l.

  residual enzymatic activity/normal value. Drastic mutations are in bold, mutations with moderate effect are in normal character and mutations without effect are in italics. The c.824C>A, p.T275N mutation is underlined.

Figure 1 :

 1 Figure 1: proline oxidase activity of PRODH alleles

Table 1 : Frequency of missense mutations in controls

 1 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

	PRODH (NM_016335)
	Variant	Exon	Genotypes (%)		Alleles (%)
	c.23C>T, p.P8L	2	PP (100)	PL (0)	LL (0)	P (100)	L (0)
	c.32G>C, p.R11P	2	RR (100)	RP (0)	PP (0)	R (100)	P (0)
	c. 56C>A, p.Q19P, rs2008720	2	QQ (26) QP (51,5) PP (22,5)	Q (52)	P (48)
	c.88C>T, p.P30S, rs3815655	2	PP (82)	PS (18)	SS (0)	P (91)	S (9)
	c.172G>A, p.A58T	2	AA (95)	AT (4)	TT (1)	A (97)	T (3)
	c.553T>C, p.R185W, rs4819756	5	RR (43) RW (41) WW (16) R (63,5) W (36,5)
	c.824C>A, p.T275N, rs5747933	7	TT (93)	TN (7)	NN (0)	T (97)	N (3)
	c.1217C>T, p.P406L, rs3970555	11	PP (97)	PL (3)	LL (0)	P (98,5)	L (1,5)
	c.1279G>A, p.V427M, rs2238731	12 VV (100) VM (0)	MM (0)	V (100)	M (0)
	c.1292G>A, p.R431H, rs2904552	12	RR (78)	RH (18)	HH (4)	R (87)	H (13)
	c.1322T>C, p.L441P, rs2904551	12	LL (98)	LP (2)	PP (0)	L (99)	P (1)
	c.1331G>A, p.G444D	12 GG (100) GD (0)	DD (0)	G (100)	D (0)
	c.1357C>T, p.R453C, rs3970559	12	RR (96)	RC (4)	CC (0)	R (98)	C (2)
	c.1397C>T, p.T466M, rs2870984	12	TT (99)	TM (1)	MM (0)	T (99,5)	M (0,5)

Table 2 : Phenotypic and genotypic features of patients with Type I hyperprolinemia a: clinical features

 2 hypertelorism, telecanthus, high palate, ligamentar hyperlaxity; ++ frontal bossing, thin lips, syndactyly 2 rd -3 rd toes; +++ high forehead, frontal bossing, micrognathism, thin upper lip, short nose, short stature , microcephaly -2.5 SD; ++++ short nose with anteverted nostrils, prominent ridging of the metopic suture, long and flat philtrum and thin upper lip, small chin, hypertelorism, strabismus.Drastic mutations are in bold, mutations with moderate effect are in normal character and mutations without effect are in italics. The c.824C>A, p.T275N mutation is underlined.

	F: female; M: male; age (years); #: born from consanguineous parents; * corpus callosum
	agenesis, symmetrical temporal lobe hypoplasia, thalamus fusion; ** right opercular dysplasia
	MRI: Magnetic Resonance Imaging; aCGH: array Comparative Genomic Hybridization
	AF: autistic features; AP: atypical psychosis; At: ataxia; Au: autism; D: dyslexia; E: epilepsy;
	LD: learning disorder; MR: mental retardation; PE: pharmacoresistant epilepsy; PMD:
	Psychomotor delay.

+ b: molecular features PRODH (NM_016335); WT: wild type; del: deletion Fasting abnormal plasma proline values: age >18 years: males > 377 µmol/l, females > 316 µmol/l; 5 years>age <18 years: > 270 µmol/l; age < 5 years: > 235 µmol/l. PREA: predicted residual enzymatic activity/normal value.

Figure 1: proline oxidase activity of PRODH alleles

	Table 2b
	Plasma proline PRODH (NM_016335)				PREA
	Subject The thin vertical lines indicate standard deviation. (µmol/l) Genotype			(%)
	WT: wild type
		200
		150
	% WT	50 100
		0	WT	F r p.P8L p.R11P o	p.P30S	p.A58T Alleles	p.T275N	p.G444D p.[Q19P; P30S] p.[Q19P; A58T]
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Supp Table S1: proline oxidase activity of previously tested PRODH alleles

  [START_REF] Bender | Functional consequences of prodh missense mutations[END_REF]] PRODH (NM_016335)

	Alleles	%WT activity
	p.Q19P	65
	p.A167V	65
	p.R185W	55
	p.R185Q	100
	p.L289M	85
	p.P406L	12
	p.D426N	40
	p.V427M	45
	p.R431H	65
	p.L441P	0
	p.R453C	8
	p.A455S	90
	p.T466M	18
	p.A472T	72
	p.Q521E

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Acknowledgements

AG received a fellowship from Region Haute Normandie. We thank Dr Pascaline Gaildrat for the splicing assay monitoring. We thank the patients and their families for their participation.