Detection of Unfolded Cellular Proteins Using Nanochannel Arrays with Probe‐Functionalized Outer Surfaces

Abstract

Nanochannel technology has emerged as a powerful tool for label‐free and highly sensitive detection of protein folding/unfolding status. However, utilizing the inner wall of nanochannel array may cause multiple events even for proteins with the same conformation, posing challenges for accurate identification. Herein, we present a platform to detect unfolded proteins through electrical and optical signals using nanochannel array with outer‐surface probes. The detection principle relies on the specific binding between the maleimide groups in outer‐surface probes and the protein cysteine thiols that induce changes in the ionic current and fluorescence intensity responses of the nanochannel array. By taking advantage of this mechanism, the platform has the ability to differentiate folded and unfolded state of proteins based on the exposure of a single cysteine thiol group. The integration of these two signals enhances the reliability and sensitivity of the identification of unfolded protein states and enables the distinction between normal cells and Huntington's disease mutant cells. This study provides an effective approach for the precise analysis of proteins with distinct conformations and holds promise for facilitating the diagnoses of protein conformation‐related diseases.

Unfolded Protein-Based Sandwich AIE Probe Imparts High Fluorescent Contrast for Pan-Cancer Surgical Navigation

“…Unfolded proteins are intermediates in the process of protein synthesis that fold into stable proteins in the ER through reactions between sulfhydryl groups. According to the previous literature, 35 bovine serum albumin (BSA) as a classical model was employed to explore the responsiveness of MAT to sulfhydryl groups of unfolded proteins. Circular dichroism (CD) measurements confirmed that the different states of BSA were successfully constructed (Figures S19 and S20).…”

“…High proliferation is an essential feature of malignant cancers, which means that cancer cells need to synthesize more proteins to accomplish cell expansion, and naturally, there are more unfolded proteins as universal markers within the endoplasmic reticulum of cancer cells. − Aggregation-induced emission (AIE) luminogen with easy functionalization and high resistance to photobleaching has potential applications in the design of both targeted imaging probes and activation imaging probes. − In previous studies, the detection of unfolded proteins in cells was achieved using maleimide (MI) and AIE luminogen, but this probe with single activation could not be used to discriminate normal cells from cancer cells because of the low signal-to-noise ratio (SNR) (Scheme A). Combining targeting and activation capabilities within the single fluorescent probe can increase specificity and SNR.…”

Outer-Surface Functionalized Solid-State Nanochannels for Enhanced Sensing Properties: Progress and Perspective

“…Previous research has primarily concentrated on assessing the contribution of the inner-wall probes (PIW), often neglecting the potential impact of those on the outer surface. Recently, it has been demonstrated that analytes can also be detected when they approach the outer surface of solid-state nanochannels, specifically binding with the POS, causing changes in the current signals. − This approach allows for detection without the need for analytes to pass through solid-state nanochannels. Generally, nanochannels adorned with POS are commonly subjected to covalent bonding for modification.…”

“…In addition to the ions and small molecules mentioned above, solid-state nanochannels with outer-surface modified probes also facilitate direct and in situ sensitive detection of larger biomarkers including proteins, viruses, and cells. In 2023, Qiao et al used the relatively free space on the outer surface of nanochannel arrays for the detection of protein conformation . In Figure B, a platform employing nanochannel arrays and protein origami structures for the detection of unfolded proteins through both electrical and optical signals was realized.…”

“…In 2023, Qiao et al used the relatively free space on the outer surface of nanochannel arrays for the detection of protein conformation. 22 In Figure 3B, a platform employing nanochannel arrays and protein origami structures for the detection of unfolded proteins through both electrical and optical signals was realized. The detection principle hinged on the targeted interaction between maleimide groups in the POS and cysteine thiols in the protein, leading to alterations in the ion current and fluorescence intensity responses of the nanochannel array.…”