BACKGROUND

There is interest, and practical value, in utilizing quantitative low-volume sample techniques in mass spectrometry (MS) imaging and microsampling applications. The analysis of low-volume samples provides valuable insight into complex biological systems. However, the proteomic and metabolomic analysis of low-volume samples remains challenging due to the lack of simple, efficient, and reproducible microsampling techniques.

Efforts have been made to improve the sensitivity and throughput of low quantity sample analysis in MS-based omics including the development of specialized sample preparation devices, high-resolution separation methods, efficient spray-MS interfaces (i.e., novel ambient ionization techniques), and advancements in MS instrumentation. The sensitivity of low quantity sample analysis has been dramatically improved using these techniques but many challenges remain in quantitative low-volume sample injection and extraction.

Micropipettes have been the most commonly applied tools for the manipulation of low-volume samples. Briefly, one end of the capillary tubing is pulled to make a micropipette to aspirate samples into the capillary through a driving force. Two approaches have been applied as the driving force for the operation of micropipettes: pump-based extraction and electro-osmotic flow (EOF)-based extraction. In the pump-based micropipette extraction, a syringe is connected to a vacuum or mechanical pump to pull the sample into a capillary. Using this method, low sample injection volume can be accurately controlled. Coupling the pump-based extraction approach with offline capillary electrophoresis-MS for complex sample analysis has been used to study liveXenopus laevisand Zebrafish embryos using a pump-based micropipette method. When an EOF-based method is utilized, an electrode is inserted into a sampling capillary and current is applied to induce electro-osmotic flow. Additionally, a micropipetting method based on electro-osmotic injection was developed and applied to the analysis of a single cell. During the collection process, +2 V was maintained to prevent the sample buffer from getting into the laser-pulled tips before the cell was penetrated. Low-volume cellular contents were extracted using −2 V after penetration. This work demonstrated that EOF could be utilized as the driving force for microsampling low-volume samples such as single onion cells. Another approach utilized an electro-osmotic pump for low-volume sample extraction from a Zebrafish embryo.

Other microsampling approaches include hydrodynamic methods, fluidic force microscopy, capillary force, and electrowetting. A microfluidic chip-based platform, nanoPOTS, has been developed for low-volume sample processing in single-cell proteomics analysis. NanoPOTS reduces total processing volumes from the conventional hundreds of microliters to <200 nL within a single droplet reactor. However, a customized automated droplet-based microfluidic system must be incorporated for the low-volume manipulation in nanoPOTS.

Continuous electrospray has been demonstrated using a platinum wire inserted into a laser pulled glass capillary with pre-injected samples. Additionally, researchers have proposed a sheathless interface to generate continuous electrospray for MS detection.

As discussed above, numerous methods have been previously proposed for improving microsampling techniques. While improvements have been made, current systems lack the ability to quantitatively manipulate ultra low-volume samples. The design of simple, efficient, reproducible microsampling techniques have been a challenge. The currently available designs result in considerable sample loss; require complicated, often proprietary equipment configurations; and cannot be directly coupled with downstream separation techniques such as liquid chromatography (LC) and capillary electrophoresis (CE).

The novel electrospray-assisted device of the present disclosure addresses the deficiencies of the previously proposed ultra low-volume sample extraction methods.

