Low-flow push-pull perfusion is a sampling method that yields better spatial resolution than competitive methods like microdialysis. Because of the low flow rates used (50 nL/min) it is challenging to use this technique at high temporal resolution which requires methods of collecting, manipulating, and analyzing nanoliter samples. High temporal resolution also requires control of Taylor dispersion during sampling. To meet these challenges, push-pull perfusion was coupled with segmented flow to achieve in vivo sampling at 7 s temporal resolution at 50 nL/min flow rates. By further miniaturizing the probe inlet, sampling with 200 ms resolution at 30 nL/min (pull only) was demonstrated in vitro. Using this method, L-glutamate was monitored in the striatum of anesthetized rats. Up to 500 samples of 6 nL each were collected at 7 s intervals, segmented by an immiscible oil and stored in a capillary tube. The samples were assayed offline for L-glutamate at a rate of 15 samples/min by pumping them into a reagent addition tee fabricated from Teflon where reagents were added for a fluorescent enzyme assay. Fluorescence of the resulting plugs was monitored downstream. Microinjection of 70 mM potassium in physiological buffered saline evoked L-glutamate concentration transients that had an average maxima of 4.5 ± 1.1 μM (n = 6 animals, 3–4 injections each) and rise times of 22 ± 2 s. These results demonstrate that low-flow push-pull perfusion with segmented flow can be used for high temporal resolution chemical monitoring and in complex biological environments.

Droplet-based microfluidics is an attractive platform for screening and optimizing chemical reactions. Using this approach, it is possible to reliably manipulate nanoliter volume samples and perform operations such as reagent addition with high precision, automation, and throughput. Most studies using droplet microfluidics have relied on optical techniques to detect the reaction; however, this requires engineering color or fluorescence change into the reaction being studied. In this work, we couple electrospray ionization mass spectrometry (ESI-MS) to nanoliter scale segmented flow reactions to enable direct (label-free) analysis of reaction products. The system is applied to a screen of inhibitors for cathepsin B. In this approach, solutions of test compounds (including three known inhibitors) are arranged as an array of nanoliter droplets in a tube segmented by perfluorodecalin. The samples are pumped through a series of tees to add enzyme, substrate (peptides), and quenchant. The resulting reaction mixtures are then infused into a metal-coated, fused silica ESI emitter for MS analysis. The system has potential for high-throughput as reagent addition steps are performed at 0.7 s per sample and ESI-MS at up to 1.2 s per sample. Carryover is inconsequential in the ESI emitter and between 2 and 9% per reagent addition depending on the tee utilized. The assay was reliable with a Z-factor of ~0.8. The method required 0.8 pmol of test compound, 1.6 pmol of substrate, and 5 fmol of enzyme per reaction. Segmented flow ESI-MS allows direct, label free screening of reactions at good throughput and ultralow sample consumption.

Developing sensors for in vivo chemical monitoring is a daunting challenge. An alternative approach is to couple sampling methods with online analytical techniques; however, such approaches are generally hampered by lower temporal resolution and slow analysis. In this work, microdialysis sampling was coupled with segmented flow electrospray ionization mass spectrometry (ESI-MS) to perform in vivo chemical monitoring. Use of segmented flow to prevent Taylor dispersion of collected zones and rapid analysis with direct ESI-MS allowed 5 s temporal resolution to be achieved. The MS “sensor” was applied to monitoring acetylcholine in the brain of live rats. The detection limit of 5 nM was sufficient to monitor basal acetylcholine as well as dynamic changes elicited by microinjection of neostigmine, an inhibitor of acetycholinesterase that evoked rapid increases in acetycholine, and tetrodotoxin, a blocker of Na+ channels, that lowered the acetylcholine concentration. The versatility of the sensor was demonstrated by simultaneously monitoring metabolites and infused drugs.

An off-line in vivo neurochemical monitoring approach was developed based on collecting nanoliter microdialysate fractions as an array of "plugs" segmented by immiscible oil in a piece of Teflon tubing. The dialysis probe was integrated with the plug generator in a polydimethlysiloxane microfluidic device that could be mounted on the subject. The microfluidic device also allowed derivatization reagents to be added to the plugs for fluorescence detection of analytes. Using the device, 2 nL fractions corresponding to 1-20 ms sampling times depending upon dialysis flow rate, were collected. Because axial dispersion was prevented between them, each plug acted as a discrete sample collection vial and temporal resolution was not lost by mixing or diffusion during transport. In vitro tests of the system revealed that the temporal resolution of the system was as good as 2 s and was limited by mass transport effects within the dialysis probe. After collection of dialysate fractions, they were pumped into a glass microfluidic chip that automatically analyzed the plugs by capillary electrophoresis with laser-induced fluorescence at 50 s intervals. By using a relatively low flow rate during transfer to the chip, the temporal resolution of the samples could be preserved despite the relatively slow analysis time. The system was used to detect rapid dynamics in neuroactive amino acids evoked by microinjecting the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) or K + into the striatum of anesthetized rats. The resulted showed increases in neurotransmitter efflux that reached a peak in 20 s for PDC and 13 s for K + .

Microfabricated fluidic systems have emerged as a powerful approach for chemical analysis. Relatively unexplored is the use of microfabrication to create sampling probes. We have developed a sampling probe microfabricated in Si by bulk micromachining and lithography. The probe is 70 μm wide by 85 μm thick by 11 mm long and incorporates two buried channels that are 20 μm diameter. The tip of the probe has two 20 μm holes where fluid is ejected or collected for sampling. Utility of the probe was demonstrated by sampling from the brain of live rats. For sampling, artificial cerebral spinal fluid was infused in through one channel at 50 nL/min while sample was withdrawn at the same flow rate from the other channel. Analysis of resulting fractions collected every 20 min from the striatum of rats by liquid chromatography with mass spectrometry demonstrated reliable detection of 17 neurotransmitters and metabolites. The small probe dimensions suggest it is less perturbing to tissue and can be used to sample smaller brain nuclei than larger sampling devices, such as microdialysis probes. This sampling probe may have other applications such as sampling from cells in culture. The use of microfabrication may also enable incorporation of electrodes for electrochemical or electrophysiological recording and other channels that enable more complex sample preparation on the device.

Although populations of neurons are known to vary on the micrometer scale, little is known about whether basal concentrations of neurotransmitters also vary on this scale. We used low-flow push−pull perfusion to test if such chemical gradients exist between several small brain nuclei. A miniaturized polyimide-encased push−pull probe was developed and used to measure basal neurotransmitter spatial gradients within brain of live animals with 0.004 mm 3 resolution. We simultaneously measured dopamine (DA), norepinephrine, serotonin (5-HT), glutamate, γ-aminobutyric acid (GABA), aspartate (Asp), glycine (Gly), acetylcholine (ACh), and several neurotransmitter metabolites. Significant differences in basal concentrations between midbrain regions as little as 200 μm apart were observed. For example, dopamine in the ventral tegmental area (VTA) was 4.8 ± 1.5 nM but in the red nucleus was 0.5 ± 0.2 nM. Regions of high glutamate concentration and variability were found within the VTA of some individuals, suggesting hot spots of glutamatergic activity. Measurements were also made within the nucleus accumbens core and shell. Differences were not observed in dopamine and 5-HT in the core and shell; but their metabolites homovanillic acid (460 ± 60 nM and 130 ± 60 nM respectively) and 5-hydroxyindoleacetic acid (720 ± 200 nM and 220 ± 50 nM respectively) did differ significantly, suggesting differences in dopamine and 5-HT activity in these brain regions. Maintenance of these gradients depends upon a variety of mechanisms. Such gradients likely underlie highly localized effects of drugs and control of behavior that have been found using other techniques.

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Monoclonal antibodies must be both chemically and physically stable to be developed into safe and effective drugs. Although there has been considerable progress in separately understanding the molecular determinants of antibody chemical and physical stability, it remains poorly understood how defects in one property (e.g., chemical stability) impact the other property (e.g., physical stability). Here, we have investigated the impact of a common chemical modification (deamidation) on the physical stability of two monoclonal antibodies as a function of pH (from pH 3.8 to 7.4). Interestingly, we find that deamidation has significant, antibody-specific impacts on physical stability at low pH values that are common during antibody purification. Deamidation causes increases in self-association and/or aggregation at low pH (3.8), and a key contributor to this behavior appears to be deamidation-dependent increases in antibody hydrophobicity at low pH. Our findings highlight pH-dependent impacts of deamidation on antibody colloidal stability and aggregation, which are important to understand in order to improve the development and production of potent antibody therapeutics with high chemical and physical stabilities.