2.1 Dynamic Sampling Platform Design & Operation
The Dynamic Sampling Platform (DSP) for ESI-MS incorporates aspects of the previously reported Dynamic Mass Spectrometry Probe (DMSP) to create a system for direct-from-culture monitoring. Full details regarding DSP microfabrication, operation, sample treatment efficiency, and comparison to other technologies for real-time ESI-MS have been described elsewhere.19-21 As shown in Figure 2, the DSP includes 1) a spatially resolved sampling interface for sterile, direct-from-culture media uptake, 2) an optimized  “cross flow” sample treatment mass exchanger for inline sample preparation (see supplementary information for details) and 3) an inline ESI emitter for online MS analysis. The DSP is a microfabricated mass exchanger with an integrated 360 μm OD, 50 μm ID PEEK (Upchurch Scientific) inlet for sample intake (probing tip) and a fused silica nanoESI emitter with a 360 μm OD and a 75 μm ID tapered to a 30 μm emitter (New Objective) outlet for inline electrospray ionization of the sample analytes for MS analysis. The device is enclosed in a sealed fluidic package (Fig. 2) which allows for easy integration with MS. The package is designed for the introduction of high flow rate (50 mL/hr) conditioning flow to the mass exchanger section of the DSP, which is an important aspect enabling rapid molecular separation.
Sample is extracted from the cell culture and injected into the microfabricated mass exchanger sample channel at a flow rate of 30 μL/hr. The sample channel is fluidically coupled to the high flow rate conditioning channel through a thin (~5 μm) nanoporous alumina membrane. The pore size (50 nm) impedes the transport of larger biomolecules of interest while allowing smaller molecules to diffuse rapidly into the conditioner channel. Simultaneously, chemicals which have been shown to enhance ESI-MS performance (acetic acid or AA, and 3-nitrobenzyl alcohol or m-NBA) are injected into the sample channel to improve ionization and aid in MS detection of biomolecules.19, 20
 The total volume of the mass exchanger is ~20 nL, the ESI emitter ~200 nL, and the PEEK tubing ~250 nL. Given its microfluidic design, DSP is remarkably fast: at a sample flow rate of 30 μL/hr the sample transmission time is ~1 minute. Direct-from-culture sampling was achieved through a sterilized (via autoclaving) ~10 cm length of 360 μm OD, 50 μm ID PEEK tubing (IDEX) submerged directly into the cell culture. A 1 μL sample was drawn into the sampling port at 50 μL/hr and then infused through the DSP system at 30 μL/hr. Two sampling methodologies were employed with identical results: the sampling port was either attached to a switching valve to allow for switching between sample uptake and infusion (as shown in Figure 2) or attached to the DSP system via quick disconnect fitting for infusion. In both cases an identical volume was sampled and then infused through the DSP system. The integrated capillary probe enabled precise control of the inlet positioning ~50 μm to the cell surface (monitored optically via Dino-lite AM7515MT4A microscope). This small volume extraction approach ensures that only the local microenvironment is sampled and allows for more frequent sampling without affecting culture viability by removing excessive amounts of media.