Capture, Enrichment, and Mass Spectrometric Detection of Low‐Molecular‐Weight Biomarkers with Nanoporous Silicon Microparticles

Abstract

Mining the disease information contained in the low-molecular-weight range of a proteomic profile is becoming of increasing interest in cancer research. This work evaluates the ability of nanoporous silicon microparticles (NPSMPs) to capture, enrich, protect, and detect low-molecular-weight peptides (LMWPs) sieved from a pool of highly abundant plasma proteins. The average pore size and porosity of NPSMPs are controlled by the electrochemical preparation conditions, and the critical parameters for admission or exclusion of protein with a definite molecular weight are determined by reflectometric-interference Fourier transform spectroscopy (RIFTS). Sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of the proteins captured by the NPSMPs show that the chemical nature of the NPSMPs surface and the solution pH also play vital roles in determining the affinity of NPSMPs for target analytes. It is found that carboxyl-terminated porous microparticles with a porosity of 26% (pore diameter around 9.0 nm) specifically fractionate, enrich and protect LMWPs sieved from either simulated samples or human serum samples. Moreover, NPSMPs containing captured peptides can be directly spotted onto a MALDI plate. When placed in a conventional MALDI matrix, laser irradiation of the particles results in the release of the target peptides confined in the nanopores, which are then ionized and detected in the MALDI experiment. As a proof-of-principle test case, mass spectra of NPSMPs prepared using serum from colorectal cancer patients and from control patients can be clearly distinguished by statistical analysis. The work demonstrates the utility of the method for discovery of biomarkers in the untapped LMWP fraction of human serum, which can be of significant value in the early diagnosis and management of diseases.

Cancer biomarker detection: recent achievements and challenges

“…136 Another mass spectrometric sensor was fabricated by Wu's group using nanoporous silicon microparticles to capture, enrich, protect, and detect low-molecularweight peptides sieved from a pool of highly abundant plasma proteins in a colorectal cancer patient's serum. 137 Some magnetic and mechanical assays have also been used for cancer protein biomarker evaluation. [138][139][140][141] However, these techniques all require expensive equipment, sophisticated instrumentation, and specially trained technical personnel, which goes against the basic requirements of point-of-care testing that instead requires compact, robust, and inexpensive instruments.…”

Absolute quantification of DcR3 and GDF15 from human serum by LC‐ESI MS

“…In most cases, a combination of techniques is used. Recently, more unconventional techniques to purify and enrich serum proteins prior to MS detection were described, including the use of nanoporous silicon microparticles or mesoporous silica chips .…”

Storage of serum peptide information in nanoporous silicon microparticles

“…We have previously found that nanoporous Si microparticles (NPSMPs) with a suitable pore size could not only capture LMWPs from serum samples, but also effectively exclude proteases (such as trypsin) and protect the captured peptides from degradation. 8 The goal of this study is to further elucidate, with the assistance of this nanotechnology, whether the peptide information in the original serum sample can be retained under different storage conditions.…”

“…Fourier transform reflective interferometric spectroscopy was employed for establishing the relationship between cut-off M W and critical porosity of porous Si. 8 The surface of NPSMPs was modified with undecylenic acid, which can improve the stability of NPSMPs, as well as their affinity for peptides. Compared with nanoporous silica particles, 9 mesoporous silica chips or films, 10 mesoporous carbon, 11 and smart hydrogels 12 that have been employed for capturing LMWPs from serum samples, the pore diameter of pSi can be precisely tailored by electrochemical etching parameters.…”

“…Therefore, the protein-laden microparticles can be directly spotted on a MALDI plate for mass spectroscopic analysis. 8 This nanotechnology provides a powerful tool that may accelerate the peptidomic research. In the present work, the capability of NPSMPs for storing serum peptide information was evaluated.…”