Cascade Reaction-Based, Near-Infrared Multiphoton Fluorescent Probe for the Selective Detection of Cysteine.

The ability to detect and visualize cellular events and their associated target biological analytes through use of cell-permeable profluorogenic probes is dependent on the availability of activatable probes that respond rapidly and selectively to target analytes by production of fluorescent reporting molecules whose excitation and emission energies span a broad range. Herein is described a new probe, DCM-Cys, that preferentially reacts with cysteine to form a dicyanomethylene-4H-pyran (DCM) reporter whose red-energy fluorescence can be stimulated by two-photon, near-infrared excitation so as to provide visualization of cysteine presence inside living human cells with a high signal-to-background ratio. These aforementioned characteristics and the ability of DCM-Cys to provide selective, nanomolar-level in vitro cysteine detection, as demonstrated by its lack of significant response to other thiols and potential interfering agents from biological environments, are attributed to the molecular designs of the DCM-Cys probe and DCM reporter. Attachment of an acryl moiety to the DCM reporter via a self-eliminating, electron-withdrawing benzyl alcohol-carbamate linker offers a probe having selective, sensitive reaction with cysteine to rapidly produce a reporter whose energies of excitation and emission (λabsreport = 480 nm, λemisreport = 640 nm) are red-shifted from those of the DCM-Cys probe (λabsprobe = 440 nm, λemisprobe = 550 nm), thereby leading to low background signal from abundant probe and a large signal from the resulting reporter of cysteine presence.

Oxidoreductase-Facilitated Visualization and Detection of Human Cancer Cells.

Achieving highly selective and sensitive detection/visualization of intracellular biological events through the use of cell-penetrable, bioanalyte-activatable, turn-on probes is dependent on the presence of specific event-linked cellular biomarkers, if and only if there exist activatable probes that appropriately respond to the biomarker analyte. Here is described the evaluation of, and use in cellular imaging studies, a previously undisclosed naphthalimide probe QMeNN, whose fluorescence is deactivated by photoinduced electron transfer (PeT) quenching that results from the presence of a covalently linked biomarker-specific quinone trigger group. Highly selective and rapid activation of the quinone group by the human cancer tumor-linked NAD(P)H: quinone oxido-reductase isozyme 1 (hNQO1) results in fast trigger group removal to yield a highly fluorescent green-energy-range reporter that possesses a high molar absorptivity; there is a 136-fold increase in brightness for the enzymatically produced reporter versus probe precursor, a value 4 times greater than previously reported for the hNQO1 analyte. The novel probe is taken up and activated rapidly within only hNQO1-positive human cancer cells; addition of an hNQO1 inhibitor prevents the selective activation of the probe. Comparison of cytosolic fluorescence intensity in positive cells versus background in negative cells yields a quantitative metric (positive-to-negative ratio, PNR) for judging hNQO1 activity. We show it is possible to determine hNQO1 presence in previously studied colorectal cancer cells and the unexplored ovarian cancer cell line NIH:OVCAR-3, with respective PNR values of 926 and 34 being obtained. Even with 10 min probe incubation, ready discrimination of positive cells from negative cells is achieved. Cell viability is unaffected by probe presence, thereby highlighting the practicality of probe use in live-cell imaging applications.

Detection and cellular imaging of human cancer enzyme using a turn-on, wavelength-shiftable, self-immolative profluorophore.

A frontier area in the development of activatable (turn-on) fluorescence-based probes is that concerned with rapid and selective stimulus triggering of probe activation so as to allow for biomarker identification and cellular imaging. The work here is concerned with a cloaked fluorophore composed of a reporter whose fluorescence is efficiently quenched by it being bound to an activatable trigger group through a novel self-immolative linker. Highly selective and rapid activation of the trigger group is achieved by chemical and enzymatic means that result in activated trigger group detachment from the self-immolative linker, with the latter subsequently cleaved from the reporter autonomously, thereby unmasking intense, red-shifted fluorescence emission. To achieve this success, we used a trimethyl-locked quinone propionic acid trigger group and an N-methyl-p-aminobenzyl alcohol self-immolative linker attached to the reporter. Delineated here are the synthesis and characterization of this cloaked fluorophore and the evaluation of its triggered turning on in the presence of an up-regulated enzyme in human cancer cells, NAD(P)H: quinone oxidoreductase-1 (NQO1, DT-diaphorase, EC 1.6.99.2).

Rapid, photoinduced electron transfer-modulated, turn-on fluorescent probe for detection and cellular imaging of biologically significant thiols.

There is a very limited number of existing probes whose fluorescence is turned on in the presence of the class of biological thiols made up of glutathione, cysteine, and homocysteine. The extant probes for this class of biological thiols commonly have poor aqueous solubility and long analyte response times, and they demand a very high probe/thiol ratio for decreased time of significant reporter signal generation; knowledge regarding their selectivity with respect to other sulfur-based analytes is unclear. Described here is a previously unreported photoinduced electron-transfer-quenched probe (HMBQ-Nap 1) that offers highly selective and rapid in vitro detection of this class of biologically important thiols at low concentrations and low probe/thiol ratio, and importantly, very rapid imaging of these biological thiols in human cells.

Profluorogenic reductase substrate for rapid, selective, and sensitive visualization and detection of human cancer cells that overexpress NQO1.

Achieving the vision of identifying and quantifying cancer-related events and targets for future personalized oncology is predicated on the existence of synthetically accessible and economically viable probe molecules fully able to report the presence of these events and targets in a rapid and highly selective and sensitive fashion. Delineated here are the design and evaluation of a newly synthesized turn-on probe whose intense fluorescent reporter signature is revealed only through probe activation by a specific intracellular enzyme present in tumor cells of multiple origins. Quenching of molecular probe fluorescence is achieved through unique photoinduced electron transfer between the naphthalimide dye reporter and a covalently attached, quinone-based enzyme substrate. Fluorescence of the reporter dye is turned on by rapid removal of the quinone quencher, an event that immediately occurs only after highly selective, two-electron reduction of the sterically and conformationally restricted quinone substrate by the cancer-associated human NAD(P)H:quinone oxidoreductase isozyme 1 (hNQO1). Successes of the approach include rapid differentiation of NQO1-expressing and -nonexpressing cancer cell lines via the unaided eye, flow cytometry, fluorescence imaging, and two-photon microscopy. The potential for use of the turn-on probe in longer-term cellular studies is indicated by its lack of influence on cell viability and its in vitro stability.

Lipid nature and their influence on opening of redox-active liposomes.

The pathway for content release from reduction-sensitive liposomes based on a quinone-dioleoylphosphatidylethanolamine lipid conjugate (Q-DOPE) is outlined using results from fluorescent dye content release assays as well as single- and multiple-angle light scattering. Experimental observations are consistent with a shape/size change of the reduced liposomes prior to their aggregation, with subsequent near-quantitative content release achieved only when the lipid membrane experiences conditions favorable to a lamellar to an inverted hexagonal phase transition. Addition of poly(ethyleneglycol)-modified DOPE (PEG-DOPE) to the Q-DOPE liposomal formulation results in stabilization of the lipid bilayer, whereas incorporation of DOPE yields faster content release. At high DOPE concentrations, DOPE/PEG-DOPE/Q-DOPE liposomes exhibit larger content release, indicating a change in pathway for content release. The outcomes here provide a better understanding of the underlying principles of triggered liposomal content release and the potential utility of specific lipid properties for the rational design of drug delivery systems based on the novel Q-DOPE lipid.

Release rates of liposomal contents are controlled by kosmotropes and chaotropes.

Contents release from redox-responsive liposomes is anion-specific. Liposomal contents release is initiated by the contact of apposed liposome bilayers having in their outer leaflet 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), whose presence is due to the redox-stimulated removal of a quinone propionic acid protecting group (Q) from Q-DOPE lipids. Contents release occurs upon the phase transition of DOPE from its lamellar liquid-crystalline phase (Lα) to its hexagonal-II inverted micelle (HII) phase. Contents release is slower in the presence of weakly hydrated chaotropic anions versus highly hydrated kosmotropic anions and is attributed to ion accumulation near the zwitterionic DOPE headgroups, in turn altering the headgroup hydration, as indicated by the Lα → HII phase transition temperature, TH, for DOPE. The results are significant, not only for mechanistic aspects of liposome contents release in DOPE-based systems but also for drug delivery applications wherein exist at drug targeting sites variations in the type and concentration of ions and neutral species.

Human NAD(P)H:quinone oxidoreductase type I (hNQO1) activation of quinone propionic acid trigger groups.

NAD(P)H:quinone oxidoreductase type I (NQO1) is a target enzyme for triggered delivery of drugs at inflamed tissue and tumor sites, particularly those that challenge traditional therapies. Prodrugs, macromolecules, and molecular assemblies possessing trigger groups that can be cleaved by environmental stimuli are vehicles with the potential to yield active drug only at prescribed sites. Furthermore, quinone propionic acids (QPAs) covalently attached to prodrugs or liposome surfaces can be removed by application of a reductive trigger stimulus, such as that from NQO1; their rates of reductive activation should be tunable via QPA structure. We explored in detail the recombinant human NAD(P)H:quinone oxidoreductase type I (rhNQO1)-catalyzed NADH reduction of a family of substituted QPAs and obtained high precision kinetic parameters. It is found that small changes in QPA structure-in particular, single atom and function group substitutions on the quinone ring at R(1)-lead to significant impacts on the Michaelis constant (K(m)), maximum velocity (V(max)), catalytic constant (k(cat)), and catalytic efficiency (k(cat)/K(m)). Molecular docking simulations demonstrate that alterations in QPA structure result in large changes in QPA alignment and placement with respect to the flavin isoalloxazine ring in the active site of rhNQO1; a qualitative relationship exists between the kinetic parameters and the depth of QPA penetration into the rhNQO1 active site. From a quantitative perspective, a very good correlation is observed between log(k(cat)/K(m)) and the molecular-docking-derived distance between the flavin hydride donor site and quinone hydride acceptor site in the QPAs, an observation that is in agreement with developing theories. The comprehensive kinetic and molecular modeling knowledge obtained for the interaction of recombinant human NQO1 with the quinone propionic acid analogues provides insight into the design and implementation of the QPA trigger groups for drug delivery applications.

Aqueous-based initiator attachment and ATRP grafting of polymer brushes from poly(methyl methacrylate) substrates.

Many polymers, such as PMMA, are very susceptible to swelling or dissolution by organic solvents. Growing covalently attached polymer brushes from these surfaces by atom-transfer radical polymerization (ATRP) is challenging because of the typical requirement of organic solvent for initiator immobilization. We report an unprecedented, aqueous-based route to graft poly(N-isopropylacrylamide), PNIPAAm, from poly(methyl methacrylate), PMMA, surfaces by ATRP, wherein the underlying PMMA is unaffected. Successful attachment of the ATRP initiator, N-hydroxysuccinimidyl-2-bromo-2-methylpropionate, on amine-bearing PMMA surfaces was confirmed by XPS. From this surface-immobilized initiator, thermoresponsive PNIPAAm brushes were grown by aqueous ATRP to yield optically transparent PNIPAAm-grafted PMMA surfaces. This procedure is valuable, as it can be applied for the aqueous-based covalent attachment of ATRP initiator on any amine-functionalized surface, with subsequent polymerization of a variety of monomers.

Surface modification of droplet polymeric microfluidic devices for the stable and continuous generation of aqueous droplets.

Droplet microfluidics performed in poly(methyl methacrylate) (PMMA) microfluidic devices resulted in significant wall wetting by water droplets formed in a liquid-liquid segmented flow when using a hydrophobic carrier fluid such as perfluorotripropylamine (FC-3283). This wall wetting led to water droplets with nonuniform sizes that were often trapped on the wall surfaces, leading to unstable and poorly controlled liquid-liquid segmented flow. To circumvent this problem, we developed a two-step procedure to hydrophobically modify the surfaces of PMMA and other thermoplastic materials commonly used to make microfluidic devices. The surface-modification route involved the introduction of hydroxyl groups by oxygen plasma treatment of the polymer surface followed by a solution-phase reaction with heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane dissolved in fluorocarbon solvent FC-3283. This procedure was found to be useful for the modification of PMMA and other thermoplastic surfaces, including polycyclic olefin copolymer (COC) and polycarbonate (PC). Angle-resolved X-ray photoelectron spectroscopy indicated that the fluorination of these polymers took place with high surface selectivity. This procedure was used to modify the surface of a PMMA droplet microfluidic device (DMFD) and was shown to be useful in reducing the wetting problem during the generation of aqueous droplets in a perfluorotripropylamine (FC-3283) carrier fluid and could generate stable segmented flows for hours of operation. In the case of PMMA DMFD, oxygen plasma treatment was carried out after the PMMA cover plate was thermally fusion bonded to the PMMA microfluidic chip. Because the appended chemistry to the channel wall created a hydrophobic surface, it will accommodate the use of other carrier fluids that are hydrophobic as well, such as hexadecane or mineral oils.

Influence of Temperature on Molecular Adsorption and Transport at Liposome Surfaces Studied by Molecular Dynamics Simulations and Second Harmonic Generation Spectroscopy.

A fundamental understanding of the kinetics and thermodynamics of chemical interactions at the phospholipid bilayer interface is crucial for developing potential drug-delivery applications. Here we use molecular dynamics (MD) simulations and surface-sensitive second harmonic generation (SHG) spectroscopy to study the molecular adsorption and transport of a small organic cation, malachite green (MG), at the surface of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) liposomes in water at different temperatures. The temperature-dependent adsorption isotherms, obtained by SHG measurements, provide information on adsorbate concentration, free energy of adsorption, and associated changes in enthalpy and entropy, showing that the adsorption process is exothermic, resulting in increased overall entropy. Additionally, the molecular transport kinetics are found to be more rapid under higher temperatures. Corresponding MD simulations are used to calculate the free energy profiles of the adsorption and the molecular orientation distributions of MG at different temperatures, showing excellent agreement with the experimental results.

Assessment of glycoprotein interactions with 4-[(2-aminoethyl)carbamoyl]phenylboronic acid surfaces using surface plasmon resonance spectroscopy.

Reported here are analyses of the interactions between a select group of solution-phase glycoproteins and a unique boronic acid capture surface. The boronic acid derivative, 4-[(2-aminoethyl)carbamoyl]phenylboronic acid, AECPBA, was synthesized and then immobilized on carboxymethyl dextran surfaces using simple coupling methods. From surface plasmon resonance spectroscopy responses, it is found that model glycoproteins interact strongly with the AECPBA surface and subsequently can be readily released from the AECPBA surface using borate buffer. A striking difference between the glycoproteins fetuin and asialofetuin (desialylated fetuin), in terms of glycoprotein binding to the AECPBA surface, indicates that the interaction of glycoproteins with the immobilized AECPBA is dictated by the terminal saccharide of the heteroglycan chain. Surprisingly, secondary interactions of glycosylated and nonglycosylated proteins with the carboxymethyl dextran hydrogel matrix are observed. Importantly, it is demonstrated that use of tris(hydroxymethyl)aminomethane buffer allows for decreased secondary interactions of nonglycosylated proteins on the AECPBA/dextran surface, as noted with the model protein ExtrAvidin.

Reversibility of beta-amyloid self-assembly: effects of pH and added salts assessed by fluorescence photobleaching recovery.

The 40-residue peptide isoform beta-amyloid (Abeta(1-40)) is associated with Alzheimer's disease. Although found in the tangles and fibrous mats that characterize the brain in advanced stages of the disease, the toxic form of Abeta is believed to be oligomers or "protofibrils". Characterization of these fairly small structures in solution, especially in the presence of the much larger assemblies they also form, is a daunting task. Additionally, little is known about the rate of Abeta assembly or whether it can be triggered easily. Perhaps most importantly, the conditions for reversing assembly are not fully understood. Fluorescence photobleaching with modulation detection of the recovery profile is a sensitive and materials-efficient way to measure diffusers over a wide range of hydrodynamic sizes. The method does require attachment of a fluorescent label. Experiments to validate the use of 5-carboxyfluorescein-labeled Abeta(1-40) as a representative of the unlabeled, naturally occurring material included variation of photobleaching time and mixture of labeled and unlabeled materials. A dialysis cell facilitated rapid in situ changes in pH and salt conditions. Multiple steps and complex protocols can be explored with relative ease. Oligomeric aggregates were found by fluorescence photobleaching recovery to respond readily to pH and salt conditions. Changing these external cues leads to formation or disassembly of aggregates smaller than 100 nm within minutes.

Size-selective synthesis of immobilized copper oxide nanoclusters on silica.

We report a straightforward route for preparing bulk quantities of size-controlled and low size dispersity copper oxide nanoclusters on amorphous silica. Adsorption of the copper-dendrimer complex on the silica surface minimizes aggregation, which results in previously unachieved low size dispersity of the nanoclusters. Copper oxide nanoclusters with mean diameters of 1-5 nm with size dispersities of only 8-15% were prepared by calcination of silica impregnated with Cu(II)-poly(propylene imine) dendrimer complexes of varying stoichiometry. The size and size distribution of the copper oxide nanoparticles are tunably controlled by the ratio of the Cu(II) to the terminal primary amines in the copper-dendrimer complex, DAB-Am n -Cu(II) x , the surface coverage of the DAB-Am n -Cu(II) x , and the impregnation procedure. This method is anticipated to be useful in the preparation of other metal oxide nanoparticles, e.g., Ni and Fe, and with other oxide substrates.

Molecular Adsorption and Transport at Liposome Surfaces Studied by Molecular Dynamics Simulations and Second Harmonic Generation Spectroscopy.

A fundamental understanding of the factors that determine the interactions with and transport of small molecules through phospholipid membranes is crucial in developing liposome-based drug delivery systems. Here we combine time-dependent second harmonic generation (SHG) measurements with molecular dynamics simulations to elucidate the events associated with adsorption and transport of the small molecular cation, malachite green isothiocyanate (MGITC), in colloidal liposomes of different compositions. The molecular transport of MGITC through the liposome bilayer is found to be more rapid in 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPG and DOPS, respectively) liposomes, while the molecular transport is slower in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. Interestingly, MGITC is observed to neither adsorb nor transport in trimethyl quinone-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (QPADOPE) liposomes due to shielding by the quinone group. The modified Langmuir adsorption isotherm model is used to determine the free energy of adsorption for MGITC, which is found to be less negative in DOPC than in DOPG and DOPS, caused by lower electrostatic interactions between the positively charged dye and the zwitterionic DOPC liposome surface. The results are compared to our previous investigations, which showed that malachite green (MG) adsorbs and transports in DOPG and DOPS liposomes but not in DOPC and QPADOPE liposomes. Molecular dynamics simulations are used to investigate the adsorption and transport properties of MG and MGITC in DOPC and DOPG liposomes using umbrella sampling to determine the free energy profiles and interfacial molecular orientations. Together, these time-resolved SHG studies and corresponding molecular dynamics simulations characterize the complicated chemical interactions at different lipid membranes to provide key molecular-level insights for potential drug delivery applications. The results also point toward understanding the role of chemical functional groups, in this case isothiocyanate, in controlling molecular adsorption at and transport through lipid bilayers.

Redox-triggered contents release from liposomes.

An exciting new direction in responsive liposome research is endogenous triggering of liposomal payload release by overexpressed enzyme activity in affected tissues and offers the unique possibility of active and site-specific release. Bringing to fruition the fully expected capabilities of this new class of triggered liposomal delivery system requires a collection of liposome systems that respond to different upregulated enzymes; however, a relatively small number currently exist. Here we show that stable, approximately 100 nm diameter liposomes can be made from previously unreported quinone-dioleoyl phosphatidylethanolamine (Q-DOPE) lipids, and complete payload release (quenched fluorescent dye) from Q-DOPE liposomes occurs upon their redox activation when the quinone headgroup possesses specific substituents. The key component of the triggerable, contents-releasing Q-DOPE liposomes is a "trimethyl-locked" quinone redox switch attached to the N-terminus of DOPE lipids that undergoes a cleavage event upon two-electron reduction. Payload release by aggregation and leakage of "uncapped" Q-DOPE liposomes is supported by results from liposomes wherein deliberate alteration of the "trimethyl-locked" switch completely deactivates the redox-destructible phenomena (liposome opening). We expect that Q-DOPE liposomes and their variants will be important in treatment of diseases with associated tissues that overexpress quinone reductases, such as cancers and inflammatory diseases, because the quinone redox switch is a known substrate for this group of reductases.

Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor.

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.

Effect of linker structure on surface density of aptamer monolayers and their corresponding protein binding efficiency.

A systematic study is reported on the effect of linker size and its chemical composition toward ligand binding to a surface-immobilized aptamer, measured using surface plasmon resonance. The results, using thrombin as the model system, showed that as the number of thymidine (T) units in the linker increases from 0 to 20 in four separate increments (T(0), T(5), T(10), T(20)), the surface density of the aptamer decreased linearly from approximately 25 to 12 pmol x cm(-2). The decrease in aptamer surface density occurred due to the increased size of the linker molecules. In addition, thrombin binding capacity was shown to increase as the linker length increased from 0 to 5 thymidine nucleotides and then decreased as the number of thymidine residues increased to 20 due to a balance between two different effects. The initial increase was due to increased access of thrombin to the aptamer as the aptamer was moved away from the surface. For linkers greater in length than T(5), the overall decrease in binding capacity was primarily due to a decrease in the surface density. Incorporation of a hexa(ethylene glycol) moiety into the linker did not affect the surface density but increased the amount of thrombin bound. In addition, the attachment of the linker at the 3'- versus the 5'-end of the aptamer resulted in increased aptamer surface density. However, monolayers formed with equal surface densities showed similar amounts of thrombin binding irrespective of the point of attachment.

Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips.

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of approximately 7.6 +/- 1.6 microg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 +/- 5%. The immobilization and purification assay using this PPC microchip could be performed within approximately 25 min as follows: (i) DNA immobilization approximately 6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption approximately 6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for 'reactivation' of the PC surface with UV light.

Integration of large-area polymer nanopillar arrays into microfluidic devices using in situ polymerization cast molding.

Presented here is a simple and robust approach for the integration of mixed-scale (nm-cm) structures into fluidic devices. We report on devices composed of large-area polymer nanopillar arrays of high aspect ratio (33-667) integrated into microfluidic channels fabricated by cast-molding polymerization of methyl methacrylate with mechanically/lithographically patterned, nanoporous aluminium oxide (AAO) templates. The microchannels containing the nanopillar arrays can be chemically functionalized and used for a variety of applications, such as separation beds or solid-phase reactors/extractors.