nL Sample Research Articles

Effect of microstructure and chemical composition on cell orientation and proliferation viability of titanium alloy processed by pulse lasers

Samples with different surface microstructures and elemental composition were fabricated by pulsed lasers with different pulse duration, including the millisecond laser remelting (RE), the bioceramic coating (BC), and nanosecond laser processing (NL) samples. The morphologies, chemical elements, phases, and valences of these samples were analyzed and characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The contact angles of the samples were measured in air and under water. Their cell activity and behavior were analyzed by cell proliferation and cytoskeleton staining experiments. The results show that the hydrophilicity and aerophobicity increase after laser treatment, and the wetting behavior of underwater bubbles is strongly related to the wettability of the sample in the air. None of the samples examined in the experiment are cytotoxic. During the initial culture, the greater unevenness of the BC and NL sample surfaces hinders the initial expansion of the cells. In contrast, the cell spreading area of the ground (G) and the RE samples is larger. The inclined sidewall in the V-shaped structure weakens the energy barrier in the groove-ridge combination (NL sample), allowing cells that rarely adhere to the sidewall to adhere successfully. In addition, the orderly distribution of adhesion sites created by laser processing further promotes cell adhesion on the inclined sidewalls. When the cells have been cultured to Day 7, the NL sample forms a clear directional distribution of cells with its unique V-shaped structure mimicking the placoid scale on the shark skin surface.

Spatial mapping and scaling of the shear-induced transformation from bicontinuous microemulsions towards lamellar structures by coupling microfluidics and SANS.

Coupling microfluidics and small-angle neutron scattering (SANS), we investigate the influence of shear flow on a model bicontinuous microemulsion of D2O/n-octane/C10E4, examining the role of membrane volume fraction in the transformation towards a lamellar structure. We employ a contraction-expansion geometry with flow velocities in excess of 10 m s-1 and spatially map the microfluidic field using a small SANS beam, illuminating down to 10 nL sample volumes. The shear-induced, progressive, bicontinuous-to-lamellar transition is found to be promoted by additional extensional flow (>103 s-1), while fast relaxation kinetics (<2 ms) return the scattering pattern to isotropic shortly after the constriction. Further, increasing the domain size of the bicontinuous structure (determined by the membrane volume fraction) appears to amplify its response to shear. Hence, the structural changes within the dilute bicontinuous microemulsions simply scale with the volume fraction of the membrane. By contrast, the stronger response of the microemulsion with the smallest domain size, located near the bicontinuous/lamellar coexistence, indicates an influence of an already more ordered structure with fewer passages. Our findings provide insight into the high shear behaviour of microemulsions of both academic and industrial relevance.

Evaluation of on-line solid-phase extraction capillary electrophoresis–mass spectrometry with a nanoliter valve for the analysis of peptide biomarkers

On-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) is a powerful technique for high throughput sample clean-up and analyte preconcentration, separation, detection, and characterization. The most typical design due to its simplicity and low cost is unidirectional SPE-CE-MS. However, in this configuration, the sample volumes introduced by pressure depend on the dimensions of the separation capillary and some matrix components could be irreversibly adsorbed in its inner walls. Furthermore, in many cases, the requirements of on-line preconcentration are incompatible with the background electrolyte necessary for an efficient separation and sensitive MS detection. Here, we present SPE-CE-MS with a nanoliter valve (nvSPE-CE-MS) to overcome these drawbacks while keeping the design simple. The nvSPE-CE-MS system is operated with a single CE instrument and two capillaries for independent and orthogonal SPE preconcentration and CE separation, which are interfaced through an external and electrically isolated valve with a 20 nL sample loop. The instrumental setup is proved for the analysis of opioid and amyloid beta peptide biomarkers in standards and plasma samples. NvSPE-CE-MS allowed decreasing the limits of detection (LODs) 200 times with regard to CE-MS. Compared to unidirectional SPE-CE-MS, peak efficiencies were better and repeatabilities similar, but total analysis times longer and LODs for standards slightly higher due to the heart-cut operation and the limited volume of the valve loop. This small difference on the LODs for standards was compensated for plasma samples by the improved tolerance of nvSPE-CE-MS to complex sample matrices. In view of these results, the presented setup can be regarded as a promising versatile alternative to avoid complicated matrix samples entering the separation capillary in SPE-CE-MS.

Acoustic Droplet Ejection and Open Port Interface for Rapid Analysis of Metabolic Stability Assays

In vitro absorption, distribution, metabolism and elimination (ADME) assays are widely used for profiling compounds in pharmaceutical drug discovery programs. Many compounds are screened in metabolic stability assays, using liver microsomes as a model of intrinsic hepatic clearance. Analysis of metabolic stability assays has relied on high throughput LC-MS/MS techniques to keep up with automated assays and compound profiling needs. An experimental alternative to sample analysis via fast chromatography employs an open port interface (OPI) which dilutes and directs acoustically-ejected droplets from microtiter plates to a conventional electrospray ion source for ionization and introduction into a mass spectrometer. Metabolic stability assays of 37 commercial drug compounds using in human, dog, rat and mouse liver microsomes (LMs), were analyzed by LC-MS/MS and an experimental breadboard version of an ADE-OPI-MS/MS system. Results from the experiments comparing intrinsic clearance (CLint) generated with ADE-OPI-MS/MS vs fast LC-MS/MS for all compounds showed ≥86% of CLint values were within a factor of two with R2 ≥ 0.86 using 25 nL and 5 nL sample ejection volumes on the ADE-OPI-MS/MS instrument. Throughput with the experimental ADE-OPI-MS/MS system used in this study was more than ten-fold faster than analysis by the fast LC-MS/MS at 1.3 s/sample versus 17.2 s/sample, respectively.

High-Throughput Liquid-Liquid Extractions with Nanoliter Volumes.

Current methods for liquid-liquid extractions generally require microliter to milliliter volumes of solvents and sample, long equilibration times, and manual procedures. Extraction methods for samples in microfluidic systems have been limited as this tool is difficult to implement on the nanoliter or smaller scale. We have developed slug-flow nanoextraction (SFNE), a method based on droplet microfluidics that allows multiple liquid-liquid extractions to be performed simultaneously in a capillary tube, using only 5 nL of sample and extraction solvent per extraction. Each two-phase slug is segmented from the others by immiscible carrier fluid. We found rapid extractions (<5 s), partition coefficients matching those achieved at larger scale extractions, and potential to preconcentrate samples through volume manipulation. This method was used to accurately and rapidly determine octanol-water partition coefficients (Kow), determining identical Kow as the shake-flask method for acetaminophen, Kow = 2.48 ± 0.02. The measurement, along with calibration and 12 replicates, was complete within 5 min, consuming under 150 nL of solvent and sample. The method was also applied to extract analytes from complex biological samples prior to electrospray ionization-tandem mass spectrometry (ESI-MS/MS) at a rate of 6 s per sample, allowing for simultaneous determination of five different drugs spiked into human plasma, synthetic urine (SU), and artificial cerebral spinal fluid (aCSF) using ethyl acetate as the extraction phase. The signal-to-noise (S/N) for analytes improved up to 19-fold compared to direct ESI-MS of single-phase droplets (aqueous plugs segmented by carrier fluid), with limits of detection down to 7 nM (35 amol).

A comparative study between a miniaturized liquid junction built in a capillary gap and semi-open capillaries for nL sample infusion to mass spectrometry

This study introduces a novel design for a microfluidic element used in a high-throughput screening mass spectrometer autosampler. The original design of the sampler consists of a liquid bridge formed in a micrometer gap between two capillaries. This liquid bridge is used to receive the sample, which is later ionized and analyzed by mass spectrometry. However, this liquid bridge is difficult to establish and maintain for long time periods, making the screening of large libraries of compounds a tedious task. The improvement described here consists in replacing the liquid bridge by a single pierced capillary, called “semi-open capillary”. The fabrication of semi-open capillaries is explained. To achieve an optimum structure, two different machining methods were tested, laser ablation and electro-discharge micromachining. The advantages of this design over the previous one include reduction of the dead volume and that of the sample dilution, as well as higher stability. Characterizations of the repeatability and the limit of detection (LOD) show that the optimized semi-open capillary leads to nearly twofold lower LOD when compared to the original liquid bridge.

Microfluidic Electrochemical Devices for Biosensing

The integration of the electrochemical detection system together with microfluidic technology is an attractive choice for the construction of miniaturized components in a single platform. Microchannel networks fabricated on conductive substrates prevent environmental contaminants and require only a tiny (µL or nL) sample for electroanalysis. Microfluidics coupled electrochemical detection system is particularly advantageous compared to traditional electrochemical sensing systems due to its flexibility, rapid analysis, low fabrication costs, ease of implementation and disposability. With these electrochemical sensing platforms, biochemical assays that require complex pre-processing of biological samples can be conducted on a chip. In this review, a comprehensive overview of the basic concepts of microfluidics and its recent applications in the design of miniaturized electrochemical sensors for biosensing applications are presented.

Microscale Reversed-Phase Liquid Chromatography/Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Deep and Highly Sensitive Bottom-Up Proteomics: Identification of 7500 Proteins with Five Micrograms of an MCF7 Proteome Digest.

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has been well recognized for bottom-up proteomics. It has approached 4000-8000 protein identifications (IDs) from a human cell line, mouse brains, or Xenopus embryos via coupling with liquid chromatography (LC) prefractionation. However, at least 500 μg of complex proteome digests were required for the LC/CZE-MS/MS studies. This requirement of a large amount of initial peptide material impedes the application of CZE-MS/MS for deep bottom-up proteomics of mass-limited samples. In this work, we coupled microscale reversed-phase LC (μRPLC)-based peptide prefractionation to dynamic pH-junction-based CZE-MS/MS for deep bottom-up proteomics of the MCF7 breast cancer cell proteome starting with only 5 μg of peptides. The dynamic pH-junction-based CZE enabled a 500 nL sample injection from as low as a 1.5 μL peptide sample, using up to 33% of the available peptide material for an analysis. Two kinds of μRPLC prefractionation were investigated, C18 ZipTip and nanoflow RPLC. C18 ZipTip/CZE-MS/MS identified 4453 proteins from 5 μg of the MCF7 proteome digest and showed good qualitative and quantitative reproducibility. Nanoflow RPLC/CZE-MS/MS produced over 7500 protein IDs and nearly 60 000 peptide IDs from the 5 μg of MCF7 proteome digest. The nanoflow RPLC/CZE-MS/MS platform reduced the required amount of complex proteome digests for LC/CZE-MS/MS-based deep bottom-up proteomics by 2 orders of magnitude. Our work provides the proteomics community with a powerful tool for deep and highly sensitive proteomics.

Open nanofluidic films with rapid transport and no analyte exchange for ultra-low sample volumes.

Moving to ultra-low (<100 nL) sample volumes presents numerous challenges, many of which can be resolved by implementation of open nanofluidic films. These nanofluidic films are fabricated using a hexagonal network of gold-coated open microchannels which capture all of the following innovative advantages: (1) sample volumes of <100 nL cm-2; (2) zero analyte exchange and loss with the film materials; (3) rapid and omni-directional wicking transport of >500 nL min-1 per square of film; (4) ultra-simple roll-to-roll fabrication; (5) stable and bio-compatible super-hydrophilicity for weeks in air by peptide surface modification. Validation includes both detailed in vitro characterization and in vivo validation with sweat transport from the human skin. Sampling times (skin-to-sensor) of <3 min were achieved, setting new benchmarks for the field of wearable sweat sensing. This work addresses significant challenges for sweat biosensing, or for any other nano-liter regime (<100 nL) fluid sampling and sensing application.

Highly Sensitive Broadband Raman Sensing of Antibiotics in Step-Index Hollow-Core Photonic Crystal Fibers

Broadband fiber enhanced Raman spectroscopy (FERS) in aqueous media is introduced in elaborated hollow-core photonic crystal fibers. The high requirements on the filigree fiber structure were thoroughly analyzed. A preparation technique was developed for the precise selective filling of such high-performance sensor fibers. Broadband step-index light guidance in aqueous solutions was achieved for the first time in photonic crystal fibers across the whole visible and NIR spectral range. Thus, strong Raman signal enhancements could be demonstrated with various excitation wavelengths and for the high-wavenumber region. The novel fiber enhancement technique was exploited for low-concentration measurements of the frequently used broad-spectrum antibiotic moxifloxacin down to 1.7 μM in only a 4 nL sample volume. These unique abilities for chemical-selective, highly sensitive antibiotic monitoring will pave the way for rapid, minimally invasive analysis of antibiotic concentrations in serum and other body fluids ...

Broadband absorption spectroscopy for rapid pH measurement in small volumes using an integrated porous waveguide.

Broadband absorption spectroscopy integrated with microfluidics is widely used to measure the pH of samples available in small volumes. Single pass absorption spectroscopy, however, suffers from poor minimum detectable absorption coefficient because the optical path length is determined by the physical dimensions of microchannels. As a result, either large amounts of indicator dyes are added or instrumentally complex techniques are used to determine sample pH. This work addresses these limitations by the integration of metal-clad leaky waveguide (MCLW) with microfluidics to perform broadband absorption spectroscopy. The MCLW consisted of a porous agarose waveguide with an estimated thickness and swelling ratio values of 1.405 ± 0.009 μm and 20.96 ± 0.65% respectively coated onto a titanium layer 32.8 ± 0.43 nm thick on a glass slide. MCLW permits obtaining absorption spectra over most of the wavelength range of the visible region (i.e. 450 nm to 700 nm) with a speed of 12.5 spectra per second using 3.2 nl of sample. The minimum detectable decadic absorption coefficient measured using MCLW was 0.43 cm-1, which is intermediate between single pass absorption spectroscopy and instrumentally complex techniques such as broadband cavity enhanced absorption spectroscopy and whispering gallery mode resonators.

Capillary electrophoresis inductively coupled plasma mass spectrometry combined with metal tag for ultrasensitively determining trace saxitoxin in seafood.

As one of paralytic shellfish toxins, the saxitoxin (STX) in the aqueous environment can be accumulated by most shellfish, and thus harms human health through the food chain. Therefore, it is crucial to determine trace STX in seafood samples in order to ensure the safety of seafood consumption. In this study, we developed a novel indirect method for ultrasensitively determining trace STX in seafood by using CE-ICP-MS together with Eu3+ chelate labeling. We demonstrated that diethylenetriamine-N,N,N',N″,N″-pentaacetic acid (DTPA) can couple with STX and simultaneously chelate with Eu3+ to realize metallic labeling of STX, and thus realize the ultrasensitive quantification of trace STX with CE-ICP-MS. The proposed method has strong antiinterference ability, good stability, and extremely high sensitivity. It could be used to determine trace STX in seafood samples with an extremely low detection limit of 0.38 fmol (3.8×10-9 M, 100 nL sample injection) and a relative standard deviation (RSD, n = 5) <7%. The success of this study provides an alternative to precise quantification of ultra-trace STX in seafood samples, and further expands the application of ICP-MS.

Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy.

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM.

Characterization of a miniaturized liquid bridge for nL sample infusion: a comparative study of sample flush-out behavior using flow simulations and direct ESI-MS analysis

In this work we shed light on the microfluidics of a miniaturized liquid bridge that forms the central part of a so-called “capillary gap sampler,” a novel device for rapid and seamless injection of nanoliter sample volumes into an electrospray ionization mass spectrometer (ESI-MS). Parameters relevant for sample flush-out at the liquid bridge and in the spray capillary were identified by systematic variation of the capillary dimensions, the linear buffer flow rate (2.1–34 mm/s) and molecular weight of the analytes (0.5–30 kDa). We found that a reduction in capillary wall thickness by a factor of 1.6 significantly influences analyte peak shapes, leads to an inversion of the relationship between peak width and analyte molecular weight, and allows a fivefold decrease in peak width for large molecules down to 5 s. The results could be verified and explained by simulations, in which the presence of diffusion-controlled “dead zones” at the liquid bridge and dispersion in the spray tip that depend on analyte molecular weight were identified as key factors relevant for the sample flush-out process. The merging of simulations and experimental data gives useful hints toward the re-design of a spray tip as built-in ESI-MS interface for an optimized gap sampler performance.

Near real-time determination of metabolic parameters for unquenched 6-mercaptopurine and xanthine oxidase samples using capillary electrophoresis

The enzyme activity of xanthine oxidase (XO) is influenced by several environmental factors including solution conditions, storage conditions, inhibitors, other enzymes, and activators. For instance, the metabolic reaction involving XO and the pro-drug 6-mercaptopurine, a drug used in the treatment maintenance of acute lymphatic leukemia, Crohn's disease, and ulcerative colitis, is often modified through the use of inhibitors, which varies the kinetic parameters associated with this reaction. Methods that provide fast and accurate determination of these kinetic constants can help in understanding the mechanism of these reactions. Herein, sequential and time-delayed electrokinetic injections of unpurified and unquenched samples containing xanthine oxidase, 6-mercaptopurine, and the inhibitor allopurinol are evaluated using capillary electrophoresis (CE). Using progress curve analysis, the Michaelis constant, apparent Michaelis constant, and inhibition constant are estimated to be 43.8±2.0μM, 143.0±3.7μM and 13.2±1.4μM, respectively. In addition, a turnover number of 7.9±0.2min−1 is quantified. These values are consistent with some previously published values but were obtained without user intervention for reaction monitoring. This unique application of CE enzyme assays offers substantial advantages over traditional methods by determining kinetic parameters for enzymatic reactions with minimal (nL) sample volumes, short (<30min) reaction analysis times, without any sample quenching or purification, and minimal user intervention.

AMPFLUID: Aggregation Magnified Post-Assay Fluorescence for Ultrasensitive Immunodetection on Digital Microfluidics

Several strategies are currently employed to enhance the detection limit of bead-based assays, but all these approaches improve the sensitivity by varying the assay procedures chemically or biologically. In previous digital microfluidic setups for bead-based immunoassay, the magnetic beads were suspended for detection. We investigated the effect of bead aggregation in such an immunoassay system and propose the aggregation magnified post-assay fluorescence for ultrasensitive immunodetection on digital microfluidics (AMPFLUID). The detection signal and sensitivity are further enhanced even at the post-assay stage without altering the original assay protocol on employing magnetically triggered post-assay aggregation of beads in a digital microfluidic setup followed by processing of the fluorescent signal. This method is shown to enhance the fluorescent signal with increased consistency and sensitivity after appropriate charge-coupled device (CCD) calibration. This method of on-chip detection allows the fulfilment of consumption of a volume at the nanoliter level and a limit of detection in the range picogram/mL. In our sTNF–RI model immunoassay, only 2.5 nL of sample is required; a detection limit 15 pg/mL is achieved. The decreased uncertainty of the measure is indicated by the error bars and coefficient of variation.

Total cysteine and glutathione determination in hemolymph of individual adult D. melanogaster

Determination of thiols, glutathione (GSH) and cysteine (Cys) are important due to their roles in oxidative stress and aging. Oxidants such as soluble O2 and H2O2 promote oxidation of thiols to disulfide (SS) bonded dimers affecting quantitation accuracy. The method presented here reduces disulfide-bonded species followed by fluorescence labelling of the 29.5 (±18.2)nL hemolymph volumes of individual adult Drosophila Melanogaster. The availability of only tens of nanoliter (nL) samples that are also highly volume variant requires efficient sample handling to improve thiol measurements while minimizing sample dilution. The optimized method presented here utilizes defined lengths of capillaries to meter tris(2-carboxyethyl)phosphine reducing reagent and monobromobimane derivatizing reagent volumes enabling Cys and GSH quantitation with only 20-fold dilution. The nL assay developed here was optimized with respect to reagent concentrations, sample dilution, reaction times and temperatures. Separation and identification of the nL thiol mixtures were obtained with capillary electrophoresis-laser induced fluorescence. To demonstrate the capability of this method total Cys and total GSH were measured in the hemolymph collected from individual adult D. Melanogaster. The thiol measurements were used to compare a mutant fly strain with a non-functional cystine–glutamate transporter (xCT) to its background control. The mutant fly, genderblind (gb), carries a non-functional gene for a protein similar to mammalian xCT whose function is not fully understood. Average concentrations obtained for mutant and control flies are 2.19 (±0.22) and 1.94 (±0.34)mM Cys and 2.14 (±0.60) and 2.08 (±0.71)mM GSH, respectively, and are not significantly different (p>0.05). Statistical analysis showed significant differences in total GSH of males and females independent of the xCT mutation. Overall, the method demonstrates an approach for effective chemical characterization of thiols in nL sample volumes.

Microfluidic integration of wirebonded microcoils for on-chip applications in nuclear magnetic resonance

We present an integrated microfluidic device for on-chip nuclear magnetic resonance (NMR) studies of microscopic samples. The devices are fabricated by means of a MEMS compatible process, which joins the automatic wirebond winding of solenoidal microcoils and the manufacturing of a complex microfluidic network using dry-photoresist lamination. The wafer-scale cleanroom process is potentially capable of mass fabrication. Since the non-invasive NMR analysis technique is rather insensitive, particularly when microscopic sample volumes are to be investigated, we also focus on the optimization of the wirebonded microcoil for this purpose. The on-chip measurement of NMR signals from a 20 nl sample are evaluated for imaging analysis of microparticles, as well as for spectroscopy. Whereas the latter revealed that the sensitivity of the MEMS microcoil is comparable with hand-wound devices and achieves a full-width-half-maximum linewidth of 8 Hz, the imaging experiment demonstrated 10 μm isotropic spatial resolution within an experiment time of 38 min for a 3D image with a field of view of 1 mm × 1 mm × 0.5 mm (500 000 voxels).

A Kinesin Driven Enzyme Linked Immunosorbant Assay (ELISA) for Ultra Low Protein Detection Applications

Detecting low levels of protein in serum samples is critical for early disease detection. In clinical settings ELISA is the most common method of protein detection. Typical ELISA requires long incubation times, significant handling procedures, and has a detection limit that is often too high for early disease detection. Our goal is to develop a kinesin-driven ELISA platform that reduces typical ELISA incubation times, eliminates laborious rinse steps, and challenges the standard ELISA detection limit. To achieve this goal we incorporate antibody-functionalized microtubule shuttles into our previous microfluidic, kinesin-driven concentrator device (NanoLett.8:1041). These functionalized microtubules bind high densities of antibody on the microtubule surface (∼100 antibodies per μm of microtubule) while retaining motility characteristics. Theoretically, these microtubules allow for the adsorption of small amounts of analyte on the microtubule surface in small (nL) sample volumes, ideal for low detection limits in ELISA. Concentrating the fluorescently-tagged protein of interest in a 625μm−2 area increases the signal to noise ratio of the fluorescent signal leading to lower detection limits that are not restricted by fluorescent background. By incorporating these microtubules into the concentrator device, we collected data to determine the detection limit and optimize the assay conditions to create a novel ELISA. Our data suggests that integrating the developed antibody-functionalized microtubules into the concentrator device creates an ELISA platform that significantly reduces the assay time, improves the ease of use, and challenges detection limits of standard ELISA. In conclusion, by successfully powering this concentrator device via kinesin driven solely by chemical energy and integrating appropriately functionalized microtubules, we created a novel ELISA platform that does not require external pumps, power supplies, or excessive handling procedures. Furthermore, this kinesin-driven technology rivals the detection capabilities of the standard ELISA platform.

Coupling liquid chromatography/mass spectrometry detection with microfluidic droplet array for label-free enzyme inhibition assay

In this work, the combination of droplet-based microfluidics with liquid chromatography/mass spectrometry (LC/MS) was achieved, for providing a fast separation and high-information-content detection method for the analysis of nanoliter-scale droplets with complex compositions. A novel interface method was developed using an oil-covered droplet array chip to couple with an LC/MS system via a capillary sampling probe and a 4 nL injection valve without the need of a droplet extraction device. The present system can perform multistep operations including parallel enzyme inhibition reactions in nanoliter droplets, 4 nL sample injection, fast separation with capillary LC, and label-free detection with ESI-MS, and has significant flexibility in the accurate addressing and sampling of droplets of interest on demand. The system performance was evaluated using angiotensinI and angiotensinII as model samples, and the repeatabilities of peak area for angiotensinI and angiotensinII were 2.7% and 7.5% (RSD, n = 4), respectively. The present system was further applied to the screening for inhibitors of cytochrome P450 (CYP1A2) and measurement of the IC50 value of the inhibitor. The sample consumption for each droplet assay was 100 nL, which is reduced 10-100 times compared with conventional 384-multi-well plate systems usually used in high-throughput drug screening.