The development of efficient biomolecular separation and purification techniques is of critical importance in modern genomics, proteomics, and biosensing areas, primarily due to the fact that most biosamples are mixtures of high diversity and complexity. Most of existent techniques lack the capability to rapidly and selectively separate and concentrate specific target proteins from a complex biosample, and are difficult to integrate with lab-on-a-chip sensing devices. Here, we demonstrate the development of an on-chip all-SiNW filtering, selective separation, desalting, and preconcentration platform for the direct analysis of whole blood and other complex biosamples. The separation of required protein analytes from raw biosamples is first performed using a antibody-modified roughness-controlled SiNWs (silicon nanowires) forest of ultralarge binding surface area, followed by the release of target proteins in a controlled liquid media, and their subsequent detection by supersensitive SiNW-based FETs arrays fabricated on the same chip platform. Importantly, this is the first demonstration of an all-NWs device for the whole direct analysis of blood samples on a single chip, able to selectively collect and separate specific low abundant proteins, while easily removing unwanted blood components (proteins, cells) and achieving desalting effects, without the requirement of time-consuming centrifugation steps, the use of desalting or affinity columns. Futhermore, we have demonstrated the use of our nanowire forest-based separation device, integrated in a single platform with downstream SiNW-based sensors arrays, for the real-time ultrasensitive detection of protein biomarkers directly from blood samples. The whole ultrasensitive protein label-free analysis process can be practically performed in less than 10 min
Schematic illustration of the preparation and operation of the SiNW forest-based capturing device. (1) Deposition of large-scale polystyrene beads monolayer. (2) Ag-assisted chemical etching of the beads-protected surface for the formation of dense SiNW forests. (3) Chemical modification of SiNW forests with antibody receptor units. (4) Interaction of the antibody-modified SiNW forests with complex biological sample (e.g., blood). (5) Washing out step. (5) Release of the specifically bound protein molecules in a controlled liquid sample. SB term represents ‘sensing buffer’.
Representative SEM images of (A-B) As-deposited large-scale PS-beads monolayer before plasma-etching (scale bars: A, 20 μm; inset, 2 μm; B, 1 μm), (C) Plasma-etched PS-beads monolayer leading to smoothly etched beads (scale bars C, 2 μm, and inset, 1 μm), (D, E, and F) Plasma etched PS beads monolayer leading to beads elements of controlled increasing roughness (scale bars: D, 1 μm, and inset, 200 nm; E, 400 nm, and inset, 1 μm; F, 1 μm).
(A) Schematic representation of the operation of a whole-SiNW selective filtering and sensing device on a single chip platform. (B) Real time calibration sensing experiments using anti-troponin T modified SiNW FETs at different concentrations of the antigen, from 10 nM down to 50 pM, in 150 μM sensing buffer solution. (C) Real-time sensing experiments of troponin T-spiked blood samples after their complete analysis using the device in part A, as a function of elution time of the antigen protein from the SiNW forest-based capturing element into the coupled SiNW-FET sensing devices. Inset: Amount of troponin T protein released from the SiNW forest-basesd capturing device, as a function of eluting time, as detected by the coupled SiNW FET sensing arrays. SB term represents ‘sensing buffer’ baseline solution