Recent Advances in Functional Nanomaterials for Diagnostic and Sensing Using Self-Assembled Monolayers.

Functional nanomaterials have attracted attention by producing different structures in any field. These materials have several potential applications, including medicine, electronics, and energy, which provide many unique properties. These nanostructures can be synthesized using various methods, including self-assembly, which can be used for the same applications. This unique nanomaterial is increasingly being used for biological detection due to its unique optical, electrical, and mechanical properties, which provide sensitive and specific sensors for detecting biomolecules such as DNA, RNA, and proteins. This review highlights recent advances in the field and discusses the fabrication and characterization of the corresponding materials, which can be further applied in optical, magnetic, electronic, and sensor fields.

Nanoprojectile Secondary Ion Mass Spectrometry for Analysis of Extracellular Vesicles.

We describe a technique based on secondary ion mass spectrometry with nanoprojectiles (NP-SIMS) for determining the protein content of extracellular vesicles, EVs, via tagged antibodies. The technique uses individual gold nanoprojectiles (e.g., Au4004+ and Au28008+), separated in time and space, to bombard a surface. For each projectile impact (10-20 nm in diameter), the co-emitted molecules are mass analyzed and recorded as an individual mass spectrum. Examining these individual mass spectra for co-localized species allows for nanoscale mass spectrometry to be performed. The high lateral resolution of this technique is well suited for analyzing nano-objects. SIMS is generally limited to analyzing small molecules (below ∼1500 Da); therefore, we evaluated three molecules (eosin, erythrosine, and BHHTEGST) as prospective mass spectrometry tags. We tested these on a model surface comprising a mixture of all three tags conjugated to antibodies and found that NP-SIMS could detect all three tags from a single projectile impact. Applying the method, we tagged two surface proteins common in urinary EVs, CD63 and CD81, with anti-CD63-erythrosine and anti-CD81-BHHTEGST. We found that NP-SIMS could determine the relative abundance of the two proteins and required only a few hundred or thousand EVs in the analysis region to detect the presence of the tagged antibodies.

Electrochemical aptamer-based biosensor developed to monitor PSA and VEGF released by prostate cancer cells.

Early prostate cancer (PCa) diagnostic is crucial to enhance patient survival rates; besides, non-invasive platforms have been developed worldwide in order to precisely detect PCa biomarkers. Therefore, the aim of the present study is to develop a new aptamer-based biosensor through the self-assembling of thiolated aptamers for PSA and VEGF on the top of gold electrodes. This biosensor was tested in three prostate cell lines (RWPE-1, LNCaP and PC3). The results evidenced a stable and sensitive sensor presenting wide linear detection ranges (0.08-100 ng/mL for PSA and 0.15 ng-100 ng/mL for VEGF). Therefore, the aptasensor was able to detect the patterns of PSA and VEGF released in vitro by PCa cells, which gave new insights about the prostate cancer protein dynamics. Thus, it could be used as a non-invasive PCa clinical diagnosis instrument in the near future. Graphical Abstract Overview of the experimental design applied to the aptamer-based electrochemical sensor self-assembled on the thiolated hairpin structure. A filter membrane was added on top of working electrode to provide the cell-attachment surface after aptamer incubation, without compromising the aptamer layer. The pore membrane allowed target proteins to pass to the aptamer surface; the MCH backfilling avoided unspecific protein binding to the gold electrode surface.

Detection of Prostate Cancer Biomarker PCA3 by Using Aptasensors.

BACKGROUND: Prostate cancer cells have very high PCA3 messenger RNA levels, which turns them into one of the new biomarkers for prostate cancer prognosis and diagnosis. OBJECTIVE: Our goal here is to develop a new aptasensor to detect PCA3 release by the cancer cell. METHODS: DNA hairpin containing PCA3 aptamer was thiolated, conjugated to methylene blue (MB) redox probe, and immobilized on gold electrode through self-assembly to detect label-free cancer cells. RESULTS: Our data have evidenced stable and sensitive sensors presenting a wide linear detection range (0-150ng/mL). In addition, monitoring PCA3 released by different types of prostate cells can provide in-depth knowledge about prostate cancer dynamics; therefore, it is a powerful platform for earlier clinical diagnostic. The released PCA3 can vary depending on the type of adopted prostate cells. CONCLUSION: PCA3 release was monitored in a group of cells for 2 h; it showed significantly higher expression in both LNCaP and PC-3 cells. This strategy provides a unique and simple methodology to achieve more sensitive and specific PCA3 detection; thus, it emerged as a promising tool for early cost-effective diagnosis.

Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid bilayer particles secreted from various cells. EVs carry molecular information of parent cells and hold considerable promise for early disease diagnostics. This paper describes a general strategy for multiplexed immunosensing of EV surface proteins, focusing on surface markers CD63, CD81, nephrin, and podocin to prove the concept. This sensing strategy entailed functionalizing gold nanoparticles (AuNPs) with two types of antibodies and then tagging with metal ions, either Pb2+ or Cu2+. The metal ions served as redox reporters, generating unique redox peaks at -0.23 and 0.28 V (vs Ag/AgCl) during electrochemical oxidation of Pb2+ and Cu2+, respectively. Capture of EVs on the working electrode, followed by labeling with immunoprobes and square wave voltammetry, produced redox currents proportional to concentrations of EVs and levels of expression of EV surface markers. Importantly, metal-ion tagging of immunoprobes enabled detection of two EV surface markers simultaneously from the same electrode. We demonstrated dual detection of either CD63/CD81 or podocin/nephrin surface markers from urinary EVs. The NP-enabled immunoassay had a sensitivity of 2.46 × 105 particles/mL (or 40.3 pg/mL) for CD63- and 5.80 × 105 particles/mL (or 47.7 pg/mL) for CD81-expressing EVs and a linear range of four orders of magnitude. The limit of detection for podocin and nephrin was 3.1 and 3.8 pg/mL, respectively. In the future, the capacity for multiplexing may be increased by extending the repertoire of metal ions used for redox tagging of AuNPs.

A new immunoassay of hybrid nanomater conjugated to aptamers for the detection of dengue virus.

A new immunosensor using hybrid nanomaterials for the detection of dengue virus was demonstrated in this work. This immunosensor composed of nanoparticles of γ-Fe2O3(SAMN) modified with MPA- SAMN@MPA was characterized by FTIR spectroscopy, transmission electron microscopy,quartz crystal microbalance, UV-vis and LSPR technique. The binding of SAMN@MPA with AuNPs conjugated with aptamers(SAMN@MPA@AuNPs@aptamer) provides specific chemical bonds to four dengue serotypes. Colorimetric changes in the modification steps provided rapid visual detection of the virus without the use of equipment. Variations of aptamers concentrations 1.0-10.0 µM where the 3.0 µM aptamer concentration is sufficient to completely cover the surface of the modified AuNPs with an R2 value of> 0.99. This new proposed methodology presenting some advantages in relation to traditional detection methods such as time optimization and cost,can be used as a diagnostic method.

An easy way to detect dengue virus using nanoparticle-antibody conjugates.

The aim of the present research is to propose a new method based on localized surface plasmon resonance (LSPR) for fast dengue virus detection. A pool with four dengue serotypes (DENV-1, -2, -3, -4) was detected through antigen-antibody binding using gold nanoparticles (AuNPs) as signaling antibody carriers. Such result was confirmed through surface plasmon resonance (SPR), transmission electron microcopy (TEM), and dynamic light scattering (DLS) techniques. The limit of detection was calculated for TCID50 107 demonstrating a linear correlation between viral concentration and number of cells with an r2 value of > 0.993. The assay presented good sensibility and reproducibility of results and the negative controls were not mistakenly detected. This design requires no pretreatment or high trained person. In the future, it can be used in commercial antibody detection kits.