Falcon Probe: A High-Pressure and Robust Sampling Interface for Coupling Lossless Liquid Chromatography Injection with In Situ Nanoliter-Scale Sample Pretreatment.

With the increasing demand for trace sample analysis, injecting trace samples into liquid chromatography-mass spectrometry (LC-MS) systems with minimal loss has become a major challenge. Herein, we describe an in situ LC-MS analytical probe, the Falcon probe, which integrates multiple functions of high-pressure sample injection without sample loss, high-efficiency LC separation, and electrospray. The main body of the Falcon probe is made of stainless steel and fabricated by the computer numerical control (CNC) technique, which has ultrahigh mechanical strength. By coupling a nanoliter-scale droplet reactor made of polyether ether ketone (PEEK) material, the Falcon probe-based LC-MS system was capable of operating at mobile-phase pressures up to 800 bar, which is comparable to those of conventional ultraperformance liquid chromatography (UPLC) systems. Using the probe pressing microamount in situ (PPMI) injection approach, the Falcon probe-based LC-MS system showed high separation efficiency and good repeatability with relative standard deviations (RSDs) of retention time and peak area of 1.8% and 9.9%, respectively, in peptide mixture analysis (n = 6). We applied this system to the analysis of a trace amount of 200 pg of HeLa protein digest and successfully identified an average of 766 protein groups (n = 5). By combining in situ sample pretreatment at the nanoliter range, we further applied the present system in single-cell proteomic analysis, and 241 protein groups were identified in single 293 cells, which preliminarily demonstrated its potential in the analysis of trace amounts of samples with complex compositions.

A minimalist approach for generating picoliter to nanoliter droplets based on an asymmetrical beveled capillary and its application in digital PCR assay.

We developed a simple approach to form picoliter to nanoliter monodisperse droplets by controlling the interface of an asymmetrical beveled capillary (ABC), with minimalist device of a beveled capillary and a liquid driving module without the need of additional equipment or external forces. We observed an evident leap decrease effect in droplet size specially existed in a capillary with a beveled outlet interface instead of a conventional flat capillary within proper bevel angle and flow rate range, by which droplets with diameters of 2-5 times the inner diameter of the capillary could be spontaneously generated by surface tension. A preliminary theoretical explanation is given to the mechanism of droplet formation at the capillary beveled interface. Various factors affecting the droplet generation process were studied, including capillary hydrophilicity, bevel angle, beveled outlet size, and inner diameter of the capillary, and dispersed phase flow rate. In the optimized condition range, good linear relationship between the droplet volume and the capillary inner diameter (10-100 µm) were obtained, which could be used to conveniently adjust the droplet volume with an adjustable droplet volume range up to 1000 times. Two types of capillaries made of fused silica and polytetrafluoroethylene (PTFE) were adopted for droplet generation using syringe pump, pneumatic pressure or gravity for liquid driving, with the relative standard deviations of droplet volume in the range of 1%-2%. To demonstrate its feasibility, the ABC approach was applied in digital PCR assay for absolute quantification of nucleic acids and identical result as a commercial instrument was obtained. The present approach has features of simple setup, easy to build without needing special microfabrication, low cost, and convenient to use, and could provide a minimalist solution for generating droplets in routine laboratories to perform single molecule analysis, single cell analysis, high-throughput screening, biochemical assays, and chemical synthesis.