Nanowire Field-Effect Transistor Sensors

Sensitive and quantitative analysis of proteins is central to disease diagnosis, drug screening, and proteomic studies. Among recent research advances exploiting new nanomaterials for biomolecule analysis, silicon nanowires (SiNWs), which are configured as field-effect transistors (FETs), have emerged as one of the most promising and powerful platforms for label-free, real-time, and highly sensitive electrical detection of proteins as well as many other biological species. Here, we describe a detailed protocol for realizing SiNW biosensors for protein detection that includes SiNW synthesis, FET device fabrication, surface receptor functionalization, and electrical sensing measurements. Moreover, incorporating both p-type and n-type SiNWs in the same sensor array provides a unique means of internal control for sensing signal verification.

G-quadruplex DNAzyme-based chemiluminescence biosensing platform based on dual signal amplification for label-free and sensitive detection of protein

17 - Semiconductor nanowires for biosensors

Herein, a general protein conversion and analysis strategy was developed for homogeneous, label-free, and sensitive protein detection, on the basis of the affinity binding-induced Hg2+ release for protein conversion, and the succeeding Hg2+ doping-induced ZnSe quantum dot (QD) photoluminescence for signal readout. Two DNA motifs were designed, each of which was conjugated with a protein-specific recognition ligand. The mercury ions were initially introduced into one DNA motif by T-Hg2+-T interaction. The Hg2+ releasing was then accomplished after protein recognition-initiated strand exchange reaction between two DNA motifs. Then, the simultaneous incorporation of the released Hg2+ into ZnSe QD resulted in a doping-dependent fluorescence emission at 560 nm correlated with protein analysis. The protein assay is outperformed only by a simple one-step mixing operation but no separation or washing steps. Also, the use of doped QD as a fluorogenic reporter can avoid the fluorophore and/or quencher labeling, and eliminate complex DNA manipulation procedures for signal readout or amplification involved in most existing nucleic acid-based protein conversion and analysis methods. The versatile and sensitive detection toward multivalent proteins was verified with the detection limits achieved at 0.034 nM for anti-Dig antibody, 0.012 nM for streptavidin, and 0.025 nM for thrombin. Thus, it shows great promise for protein analysis to accommodate the applications in disease diagnosis, biomarker screening, and clinical medicine.

Proteins are one of the most versatile groups of molecules with vital functional roles in living systems. Their enormous diversity and structural flexibility make the detection of these molecules a challenging task. A simple and sensitive label-free protein detection method based on assembly of proteins and colloidal silver nanoparticles (AgNPs) on surfaces and surface-enhanced Raman scattering (SERS) is reported. The SERS spectra from the assembled AgNP/protein films show excellent reproducibility and high quality regardless of the proteins' charge status and size. A detection limit down to 0.5 μg/mL for three acidic proteins; BSA, catalase and pepsin, and three basic proteins; cytochrome c, avidin and lysozyme, is easily achieved. The minimum improvement in detection limit is more than 1 order of magnitude compared to the previously reported detection limits using the technique and the approach has the potential for label-free protein detection and identification.

Target-induced catalytic hairpin assembly formation of functional Y-junction DNA structures for label-free and sensitive electrochemical detection of human serum proteins

Surface-enhanced Raman scattering (SERS) is an ultrasensitive optical technique that is critical for protein detection and essential for identifying protein structure and concentrations in various biomedical and diagnostic applications. However, achieving highly sensitive and reproducible SERS signals for label-free proteins remains challenging due to their weak Raman signals and structural complexity. In this study, silver nanomushroom arrays (Ag NMAs) as SERS substrates were readily prepared and surface-engineered using a facile template-assisted micro- and nanofabrication approach. The surface of the substrate exhibits nanoscale roughness, long-range order, and hydrophilicity, enabling rapid and uniform dispersion of protein molecules. These molecules are anchored through Ag-S bonds, resulting in ultrasensitive Raman signals driven by strong electromagnetic enhancement effects. The highly ordered array structure improves signal repeatability, achieving a relative standard deviation of as low as 4.32%. Additionally, utilizing the silicon characteristic peak of the SERS substrate as an internal standard significantly reduces measurement errors, allowing for reliable and precise quantitative detection of protein molecules, with a linear correlation coefficient (R2) exceeding 0.96. Ultrasensitive SERS detection and effective protein discrimination via principal component analysis further validate the Ag NMA substrate's potential for universal trace protein detection. This study presents an advanced SERS platform for the sensitive and rapid detection of trace proteins, showcasing significant potential in pharmaceutical research, metabolic studies, diagnostic medicine, and protein engineering.

In the last decade, microcantilever biosensors have shown enormous potential for highly sensitive label-free detection of nucleic acid and proteins. Despite the enormous advances, the promise of applications of this technology in the biomedical field has been frustrated because of its low reproducibility. Here we tackle the reproducibility issue in microcantilever biosensors and provide the guidelines to minimize the deviations in the biosensor response between different assays. We use as a model system the label-free end-point detection of horseradish peroxidase. We choose the end-point detection mode because of its suitability for implementation in the clinical field that requires simplicity and point-of-care capability. Our study comprises the analysis of 1012 cantilevers with different antibody surface densities, two blocking strategies based on polyethylene-glycol (PEG) and bovine serum albumin (BSA) and stringent controls. The study reveals that the performance of the assay critically depends on both antibody surface density and blocking strategies. We find that the optimal conditions involve antibody surface densities near but below saturation and blocking with PEG. We find that the surface stress induced by the antibody-antigen binding is significantly correlated with the surface stress generated during the antibody attachment and blocking steps. The statistical correlation is harnessed to identify immobilization failure or success, and thus enhancing the specificity and sensitivity of the assay. This procedure enables achieving rates of true positives and true negatives of 90% and 91% respectively. The detection limit is of 10 ng mL(-1) (250 pM) that is similar to the detection limit obtained in our enzyme-linked immunosorbent assay (ELISA) and at least two orders of magnitude smaller than that achieved with well-established label-free biosensors such as a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) sensor.

The interplay between pH sensitivity and label-free protein detection in immunologically modified nano-scaled field-effect transistor

Ultra-sensitive and quantitative analysis of proteins, nucleic acid, virus and other biochemical species are critical technologies for effective dianosis of disease, as well as medical studies. Silicon nanowires field-effect transistor (SiNWs-FET) biosensor is one of the most promising powerful platforms for label-free, real-time, ultra-sensitive detection of analyte. Here, the working principle of SiNWs-FET biosensor and the applications of SiNWs-FET biosensors in medicine were introduced. Moreover, the methods for enhancing the sensitivity of SiNWs-FET biosensor were discussed. Lastly, the prospecting of SiNWs-FET biosensor was presented.

We have demonstrated a smart polymeric transducer and aptamer/intercalating dye system that allows the label-free detection of protein with high sensitivity and selectivity.

A novel label-free universal biosensing platform based on CRISPR/Cas12a for biomarker detection

Effective detection of biomolecules is important for biological research and medical diagnosis. We here propose a ligation-triggered and protein-assisted fluorescence anisotropy amplification platform for sensitive and selective detection of small biomolecules in a complex biological matrix. In the proposed method, in the presence of target small molecules, FAM-labeled DNA 1 and biotin-labeled DNA2 were ligated to produce an integrated DNA. As a result, taking advantage of the extraordinary strong interaction between biotin and streptavidin, we employed a novel mass amplification strategy for sensitive detection of small molecules through fluorescence anisotropy. The method could detect ATP from 0.05 to 1 μM, with a detection limit of 41 nM, and detect NAD+ from 0.01 to 1 μM, with a detection limit of 6.7 nM. Furthermore, ligase-specific dependence of different cofactors provides good selectivity for the detection platform. As a result, the new platform has a broad spectrum of applications both in bioanalysis and biomedical fields.

Microscale thermophoresis sensor for homogeneous and one-step protein detection based on dual aptamers binding induced DNA switch.

Label-free bioelectronic detection of aptamer–protein interactions