•Low pressure prefractionation separations developed for nanoliter biological samples.•Reverse phase or ion exchange prefractionation increases unique protein discovery.•Longer separation introduction to mass spectrometry further increased protein hits.•Protein hits observed include those with evidence at transcript or gene level only.•Secreted proteins are dominant class of protein hits.The analysis of blood provides in depth chemical information of physiological states of organisms. Hemolymph (blood) is the fluid in the open circulatory system of Drosophila melanogaster that is the medium for molecules regulating a wide variety of physiological activities and signaling between tissues. Adult Drosophila is typically less than 3 mm in length and, as a consequence, the available volume of hemolymph is usually less than 50 nL from individual flies. Proteomic analysis of volume-limited hemolymph is a great challenge for both sample handling and subsequent mass spectrometry characterization of this chemically diverse biological fluid with a wide dynamic range of proteins in concentrations. Less abundant proteins, in particular, could be easily lost during sample preparation or missed by current mass spectrometry methods. This article describes simple and customized RPLC column and IEX columns to prefractionate volume-limited hemolymph without excessive dilution. Step-gradient elution methods were developed and optimized to enhance the identification of novel proteins from an individual fruit fly hemolymph sample. Fractions from each step gradient was analyzed by an Agilent nano-RPLC chip column and then characterized by high mass resolution and high mass accuracy orbitrap mass spectrometry. As a result, both RPLC (11 proteins) and IEX fractionation approaches (9 proteins) identified more proteins than an unfractionated control approach with higher protein scores, emPAI values and coverage. Furthermore, a significant number of novel proteins were revealed by both RPLC and IEX fractionation methods, which were missed by unfractionated controls. The demonstration of this method establishes a means to deepen proteomic analysis to this commonly used, important biological model system.

Viscosity is an easily measured macroscopic property that provides molecular information and is widely used across the sciences and engineering. Here we report a microfluidic capillary viscometer that forms droplets from aqueous samples in an immiscible carrier phase and encodes information about sample viscosity in the droplet spacing. The device shows exceptional calibration stability, with only a 0.6% drift in calibration factor from run to run, the ability to handle aqueous and nonaqueous samples, and the ability to operate with sample volumes as low as 38 nL. Operating range for aqueous sample viscosity was characterized, and was found to be 0.96–52 cP. Operating range for aqueous shear rate was found to depend on aqueous viscosity and varied from 1.9 × 101–4.4 × 102 s–1 for high viscosity samples to 4.1 × 102–6.0 × 103 s–1 for low viscosity samples. Accuracy was tested by comparing measured viscosities of several samples including crème de menthe peppermint liquor, human urine, and baby oil to viscosities of the same samples obtained with a U-tube viscometer. The device was found to be very accurate, with differences between methods as low as 0.1%. The viscometer presented requires only a basic T junction and can utilize off-chip fluorescence to measure viscosity, which could allow for easy addition of viscometric measurement capabilities to existing droplet platforms. Furthermore, the device is capable of performing measurements on Newtonian fluids without precise control over pressures or flow rates, which significantly simplifies device operation.

•Method for highly volume variant, nL sample assay of biological relevant thiols.•Defined capillary lengths used to deliver nL sample and reagent volumes.•Optimized reagent concentrations, reaction times and temperatures for thiol assay.•Total cysteine and glutathione measured from hemolymph of individual fruit flies.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.

Brain tissue slices are a common neuroscience model that allows relatively sophisticated analysis of neuronal networks in a simplified preparation. Most experimental methodology utilizes electrophysiological tools to probe these model systems. The work here demonstrates the adaptation of low-flow push–pull perfusion sampling (LFPS) to a brain slice system. LFPS is used to sample from the hippocampus of mouse brain slices. Perfusate amino acid levels are quantified following sampling with capillary electrophoresis. Glutamate was measured from the CA1 region of the hippocampus in slices taken from a cystine-glutamate transporter deletion mutant, xCT−/−, and the background strain C57BL/6J. Sampling is performed over up to 6.5 h with standard tissue slice preparation and experimentation methods. Four amino acids were quantified to demonstrate the ability to perform LFPS and show good agreement with published literature. Perfusate glutamate levels are found to be significantly lower with xCT−/− slices (1.9(±0.5) μM) relative to controls (4.90(±1.1) μM). But, experiments with control slices show a significant decrease in glutamate over the 6 h sampling period that are not seen with xCT−/− slices. Increasing the LFPS sample collection rate during the first 90 min of sampling did not show a sampling artifact in perfusate glutamate content. Sampling immediately following slicing did not show an early increasing glutamate level that would be indicative of a significant contribution from blood or tissue damage. The data presented here show a complementarity to electrophysiological studies of tissue slices. The ability to characterize extracellular fluid chemical content with LFPS in these slices provides an alternative data stream for probing neurochemical signaling networks in brain tissue slices.

•Proteomic analysis was investigated from individual fruit fly hemolymph.•Novel proteins were identified from nL of hemolymph sample.•Microliter scale protein digestion protocol was developed for nL biosample.•NanoHPLC chip column coupled with MS was efficient for nL sample.Analysis of blood proteins holds critical promise for in depth understanding of physiological states. Protein content of hemolymph from Drosophila melanogaster is of particular analytical interest because the insect open circulatory system involves chemical signaling through the hemolymph. The challenge of working with this sample, however, is the nanoliter volumes of solution available for analysis. In this study, we developed a novel hyphenated Agilent nano-HPLC chip column-MS method to obtain proteomic information from individual fruit fly hemolymph, using a low-volume sample collection technique established previously. The total amount of individual Drosophila hemolymph protein is determined around 0.798 ± 0.251 μg/100 nL based upon a Bradford assay with BSA. Hemolymph samples around 50 nL were collected from single flies and digested using a customized micro-scale digestion protocol. Mass spectral analysis shows a total of 19 proteins were identified from the hemolymph of individual flies. Of these findings, 6 novel proteins have been identified for the first time with evidence at the translation level. Detection of 13 proteins well-known in the literature speaks to the method's validity and demonstrates the ability to reproducibly analyze volume-limited samples from individual fruit flies for protein content. This nano-scale analysis method will facilitate future study of Drosophila and lead to a more complete understanding of the physiology of the fly model system.

Droplet-based microfluidic platforms sequester nanoliter to picoliter samples in an immiscible carrier phase and have gained notoriety for their ability to be used in laboratory procedures on a miniaturized scale. Recently, droplet microfluidics has been used to prevent zone diffusion in time-resolved sample collection methods and in separation techniques. The assay of droplets remains challenging, however, because the carrier phase is often incompatible with separation techniques. In this work, we report the development of a droplet injector for capillary electrophoresis (CE) which delivers 750 pL droplets to a channel for separation while excluding the fluorous carrier phase. This design is simple compared to previous reports, consisting of only two straight channels and no additional working parts such as membranes or valves. To demonstrate a proof-of-concept and characterize performance, riboflavin was used as a biologically relevant model molecule. Droplets containing a step change in riboflavin concentration were injected and mobilized by CE. The current method is capable of riboflavin peak % relative standard deviations (RSDs) down to 4.4% and temporal resolutions down to 15 s. Human urine samples containing riboflavin and its photolysis products were successfully separated and found to be chemically compatible with the injector. Our simplified design could improve robustness and ruggedness and may allow device construction via nontraditional fabrication techniques.

Glutamate analysis is useful for the study of many neurochemical processes. In vivo sampling techniques and sample assays provide a platform for monitoring extracellular glutamate levels in the central nervous system of animal models. Low flow push–pull perfusion sampling is used here to sample from the mouse striatum to understand basal extracellular glutamate levels. The 500 nL perfusate samples are pH adjusted with minimal dilution of 1.2 fold prior to derivatization for glutamate measurements. The method optimized for glutamate labelling and measurement involves the use of capillaries for addition of 100 nL solutions to samples. Quantitation of glutamate in the derivatized samples was accomplished with capillary electrophoresis-laser induced fluorescence. Glutamate and aspartate levels were monitored in C3H/HeSnJ controls and C3H/HeSnJ-Slc7a11sut mutants to understand cystine-glutamate transporter (xCT) function. The study of basal glutamate levels used 4 groups: males and females of both control and mutant mice (each N = 5). Using this approach, extracellular glutamate levels in mouse striatum were found to range between 2.68(±0.51) μM and 1.84(±0.40) μM. As expected, extracellular glutamate was lower in the striatum of sut mutant males. Control females, however, showed lower glutamate overall and no difference between genotypes. Overall, a method of LFPS collection and CE-LIF quantitation shows promise for study of glutamate in this model system.

•In vivo sampling with inhibitor infusion to assay of nitric oxide synthase activity.•Nitric oxide metabolite, nitrate, is significantly decreased with inhibitor infusions.•Nitrate levels decrease at peripheral to, but not directly over optic nerve head.•Sub-type selective inhibitor infusions display unique nitrate decrease responses.Proteins play a variety of functional roles in tissues that underlie tissue health. The measurement of protein function is important to both understand normal and dysfunctional tissue states. Low-flow push–pull perfusion sampling (LFPS) has been used to collect submicroliter volumes of extracellular fluid which are well suited to capillary electrophoresis for compositional quantitative analysis. In this study, LFPS is used to deliver pharmacological agents to the in vivo retinal tissues at the probe sampling tip during sampling to measure protein function. Two native nitric oxide synthase enzymes were pharmacologically inhibited and the enzyme product NO metabolite, nitrate, was determined with capillary electrophoresis from the perfusates. LFPS delivered inhibitors including the non-selective N(G)-nitro-Larginine methyl ester (L-NAME), the nNOS selective 7-nitroindazole (7-NI), and eNOS N5-(1-imioethyl)-L-ornithine, dihydrochloride (L-NIO) were perfused to the sampling region either directly over a rat retina optic nerve head or 1-mm peripheral to the ONH. At the PONH, 65, 55 and 60% of baseline nitrate levels, respectively, were observed with inhibitor infusion. These are statistically significant (P < 0.05) compared to saline drug infusion. However, infusion of the inhibitors to the ONH did lead to significant nitrate concentration decreases. This data suggests that the endogenous enzymes, nNOS and eNOS, are both spatially and functionally localized to the PONH at the in vivo rat retina.

The fruit fly (Drosophila melanogaster) is an extensively used and powerful, genetic model organism. However, chemical studies using individual flies have been limited by the animal’s small size. Introduced here is a method to sample nanoliter hemolymph volumes from individual adult fruit-flies for chemical analysis. The technique results in an ability to distinguish hemolymph chemical variations with developmental stage, fly sex, and sampling conditions. Also presented is the means for two-point monitoring of hemolymph composition for individual flies.

This study investigated the effect of different sampling environments on hemolymph amino acid content of individual Drosophila melanogaster larvae. Hemolymph was collected from individual third instar larvae under cold-anesthetized, awake, and stress conditions. Qualitative and quantitative hemolymph amino acid analyses were performed via capillary electrophoresis with laser-induced fluorescence detection. The hemolymph amino acid concentrations, particularly arginine, glutamate, and taurine, changed significantly depending on the prior-to-sample-collection environments. Hemolymph amino acid analyses of six different Drosophila genotypes including two control genotypes and four mutant alleles were also carried out. Two mutant genotypes with over and under expression of a putative cystine-glutamate exchanger subunit were significantly different from each other with respect to their hemolymph glutamate, glycine, lysine, and taurine levels. Hemolymph amino acid analyses of stressed larvae of two control and two mutant genotypes indicated that behavior-related hemolymph chemical changes are also genotype dependent.

A system is presented demonstrating the high-temporal resolution coupling of low-flow push-pull perfusion sampling (LFPS) to capillary electrophoresis for the absorbance measurement of ascorbate at the rat vitreoretinal interface. This system holds all separation components at a low pressure as the means for withdrawing sample during LFPS. The system uses a flow-gated interface to directly couple the withdrawal capillary from the LFPS probe to a separation capillary and eliminates the need for any offline sample handling. The temporal resolution of the system was limited by injection time and is less than 16 s. This high temporal resolution was applied to the monitoring of in vivoascorbate levels at the rat vitreoretinal interface. Baseline concentrations of ascorbate were found to be 86 µM ± 18 µM at the vitreoretinal interface. Baseline concentrations matched well with those obtained for the postmortem bulk vitreous analysis. Upon stimulation with 145 mM K+, a maximum increase in baseline values between 32–107% for n = 3 was observed. This system demonstrates the first in vivo temporal study of ascorbate at the rat vitreoretinal interface.

The determination of the presence of nitric oxide metabolites in the rat vitreous cavity in a regioselective manner is complicated by the size and shape of the eye as well as the diffusivity of the molecule and its metabolites. In this work, in vivo low-flow push−pull perfusion sampling was utilized with a rapid capillary electrophoretic assay to monitor levels of the major NO metabolite, nitrate, at the vitreoretinal interface (VRI) of normal and aged rat models. The sampling probe tips were placed in three different positions in the posterior chamber through a 29-gauge guide needle. Sampling was performed along the VRI over the optic nerve head and regions peripheral to the optic nerve head. Additionally, samples were collected from the middle vitreous region to compare to VRI sampling. A significant (P < 0.05) difference in the perfusate nitrate concentration was observed in each location, which may be due to the source of NO production or the clearance mechanism of the molecule from the vitreous cavity. Infusion of L-NAME with physiological saline led to a significant decrease (35%) in the observed nitrate level. LFPPP was then utilized to observe nitrate levels after an average of 4.5 months of aging. A 3-fold increase in the mean level of nitrate over the optic nerve head was observed in mature animals compared to younger control animals. Precise measurement of NO metabolites along the VRI may provide insights into the function of NO in maintaining homeostatic conditions and the molecular changes at the diseased retina.

A low-volume automated injection system for the analysis of chemically complex, amino acid samples is presented. This system utilizes submicroliter sample volumes stored on a 75-μm inner diameter capillary. A pulse of positive pressure (82 kPa) is used to load nanoliter sample volumes into an in-house fabricated interface and onto a separation capillary. Residual sample solution in the interface is immediately washed away by a continuous transverse flow through the injection interface, yielding a sharp and reproducible sample plug. By performing multiple injections of a static sample, one may average the signals to yield a signal-to-noise ratio improvement of up to 4.07-fold for 20 injections compared with a theoretical maximum of a 4.47-fold improvement. Without interruption of the applied voltage, injections performed every 150 s were used to monitor the progress of the reaction of multiple amino acids with the fluorogenic dye 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde. Analysis of dialyzed clinical vitreous samples demonstrates the resolution and quantitation of arginine, lysine, leucine, glutamine, and glutamate. Observed levels are comparable with those of nonautomated injection methods and reports by others.

The detection of peptides with mass spectrometry from volume-limited biological samples is a challenging task due to low sample volume, a broad range of peptide concentrations down to trace levels, endogenous high proteins and salt levels. Previously, a microspotting method was presented for trace-level peptide detection with MALDI-MS from sub-microlitre samples with biological salt levels. However, in the presence of proteins, peptide signals are significantly reduced. This paper presents a novel dialysis device for removal of proteins from sub-microlitre samples using a semipermeable hollow fiber membrane to enhance peptide detection. A dialysis device was constructed to perform sub-microlitre dialysis to remove proteins from complex samples. Angiotensin I was used as a model peptide in the presence of 350 mg L−1 BSA prepared in physiological saline to mimic biological samples. In the absence of BSA, clear angiotensin I peaks were seen at 250 pM, yet in the presence of the BSA, 10 nM angiotensin I was barely detected. After dialysis, peak detection was improved to a 500 pM level. Protein removal and peptide recovery (approximately 66%) were determined using CE-LIF. Clinical vitreous samples as low as 200 nL were successfully dialyzed in 30 min and a 3-fold increase in peptide peaks were detected with greatly improved signals. This method is simple and can be a useful technique for trace level peptide detection from volume-limited biological samples.

The determination of nitric oxide (NO) in human vitreous samples is complicated by the relatively short half-life of the analyte and the viscous, high salt and protein biological matrix. In this work, we developed a fast (<5 min) and useful CE method to determine the stable metabolite, nitrate, from vitreous samples. This proposed method has been successfully applied to determine the nitrate levels from the vitreous humor of patients undergoing vitrectomy for a variety of conditions. A statistically significant increase (P = 0.000001) of the mean level of nitrate was observed in vitreous humor of patients with proliferative diabetic retinopathy (41.17 ± 4.09 μM, n = 27) versus controls (15.22 ± 0.86 μM, n = 35). The elevated levels of nitrate in the vitreous of patients known to have diabetic retinopathy suggests that NO is involved with the pathology of this disease.

A simple automated nanoliter scale injection device which allows for reproducible 5 nL sample injections from samples with a volume of <1 µL is successfully used for conventional capillary electrophoresis (CE) and Hadamard transform (HT) CE detection. Two standard fused silica capillaries are assembled axially through the device to function as an injection and a separation capillary. Sample solution is supplied to the injection capillary using pressure controlled with a solenoid valve. Buffer solution flows gravimetrically by the junction of the injection and separation capillaries and is also gated with a solenoid valve. Plugs of sample are pushed into the space between the injection and separation capillaries for electrokinectic injection. To evaluate the performance of the injection device, several optimizations are performed including the influence of flow rates, the injected sample volume and the control of the buffer transverse flow on the overall sensitivity. The system was then applied to HT-CE-UV detection for the signal-to-noise ratio (S/N) improvement of the nitric oxide (NO) metabolites, nitrite and nitrate. In addition, signal averaging was performed to explore the possibility of greater sensitivity enhancements compared to single injections.

This paper presents a highly efficient sample preparation technique for matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The purpose of the research is to use a conventional MALDI support to directly and conveniently detect sub-nM levels of peptides from volume-limited samples with physiological salt levels. In this new method, highly uniform matrix-nitrocellulose spots with a 500 µm diameter were conveniently generated by direct contact of a capillary tip to a stainless steel MALDI plate. An array of 50 microspots can be blotted from 1 µL matrix-nitrocellulose solution within 1 min. It was found that the addition of high concentration nitrocellulose to the α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution is critical for the formation of microspots. Samples are deposited on top of those microspots and incubated for 3 min. The CHCA-nitrocellulose surface shows a significant peptide binding capability for sub-nM levels of peptide. Restricting the matrix spot diameter to 500 µm gives an analyte enrichment effect because the peptides are confined to a small solid-phase surface area. Selective peptide binding is seen even with >0.15 M salt levels. Loading small aliquots of samples with multiple applications allows low level peptide detection down to 100 pM. Push-pull perfusates collected from the rat striatum were successfully analyzed with the microspot method.

A simple and sensitive solid-phase fluorescence immunoassay method was developed to detect peptides without separating them from a biological matrix. A near infrared fluorescence detection system was constructed for scanning analyte spots blotted onto protein binding membranes. Hydrophobic membranes were used with a modified vacuum spot blotting system to concentrate the peptide solution into a small area and the overall assay time was thus reduced by eliminating blocking steps. Both direct and indirect immunoassay methods are demonstrated; the indirect is more sensitive and features a 1 pmol detection limit of neat dynorphin A solutions. To further increase the immunoassay sensitivity, a novel capillary blotting system with hydrophilic membranes was designed where optimized sample volumes of 167 nL were deposited for each spot. The area-reduced blotting method shows a 1000-fold improved, 1.3 fmol spot−1 detection limit of a dynorphin A diluted in a buffered solution of 150 mg L−1 of casein. Low-flow push-pull perfusates with volumes of 1 µL sampled from the striatum of the rat were assayed for dynorphin A by the method of standard addition. The detection limit was estimated to be 1.9 fmol in the low-flow push-pull perfusates. These data demonstrate a solid-phase near infrared immunofluorescence strategy for the study of peptides directly blotted from chemically complex biological fluid matrices.

Glutamate analysis is useful for the study of many neurochemical processes. In vivo sampling techniques and sample assays provide a platform for monitoring extracellular glutamate levels in the central nervous system of animal models. Low flow push–pull perfusion sampling is used here to sample from the mouse striatum to understand basal extracellular glutamate levels. The 500 nL perfusate samples are pH adjusted with minimal dilution of 1.2 fold prior to derivatization for glutamate measurements. The method optimized for glutamate labelling and measurement involves the use of capillaries for addition of 100 nL solutions to samples. Quantitation of glutamate in the derivatized samples was accomplished with capillary electrophoresis-laser induced fluorescence. Glutamate and aspartate levels were monitored in C3H/HeSnJ controls and C3H/HeSnJ-Slc7a11sut mutants to understand cystine-glutamate transporter (xCT) function. The study of basal glutamate levels used 4 groups: males and females of both control and mutant mice (each N = 5). Using this approach, extracellular glutamate levels in mouse striatum were found to range between 2.68(±0.51) μM and 1.84(±0.40) μM. As expected, extracellular glutamate was lower in the striatum of sut mutant males. Control females, however, showed lower glutamate overall and no difference between genotypes. Overall, a method of LFPS collection and CE-LIF quantitation shows promise for study of glutamate in this model system.