Constructing Ultra-Strong SERS Tags in the Cellular Raman-Silent Region by Orthogonal Array Testing Strategy.

Surface-enhanced Raman spectroscopy (SERS) tags have the advantages of unique fingerprint vibration spectrum, ultranarrow spectral line widths, and weak photobleaching effect, showing great potential for bioimaging. However, SERS imaging is still hindered for further application due to its weak spontaneous Raman scattering, biomolecular signal interference, and long acquisition times. Here, we develop a novel SERS tag of the core (Au)-shell (N-doped graphene) structure (Au@NGs) with ultrastrong and stable Raman signal (2180 cm-1) in the cellular Raman-silent region (1800-2800 cm-1) through base-promoted oxidative decarboxylation of amino acids. Exploring the factors (metal salts, amino acids, catalysts, temperature, etc.) to obtain Au@NGs with the strongest Raman signal commonly requires more than 100,000 separate experiments, while that using an orthogonal array testing strategy is reduced to 56. The existence of deep charge transfer between the Au surface and C≡N-graphene is proved by theoretical calculations, which means the ultrastrong signal of Au@NGs is the joint effect of electromagnetic and chemical enhancement. The Au@NGs have a detection sensitivity down to a single-nanoparticle level, and high-speed and high-resolution cellular imaging (4453 pixels) is obtained within 10 s by global Raman imaging. The combination of Au@NGs-based tags with ultrastrong intrinsic Raman imaging capability and global imaging technology holds great promise for high-speed Raman imaging.

Proteomics Landscape Mapping of Organ-Resolved Behçet's Disease Using In-Depth Plasma Proteomics for Identifying Hyaluronic Binding Protein 2 Expression Associated With Vascular Involvement.

OBJECTIVE: This study was undertaken to elucidate the pathogenesis and heterogeneity of Behçet's disease (BD) involving different organs using in-depth proteomics to identify the biomarkers for clinical assessment and treatment of patients with BD. METHODS: We measured the expression levels of proteins in plasma samples from 98 patients with BD and from 31 healthy controls using our in-depth proteomics platform with a data-independent acquisition mass spectrometer and antibody microarray. We performed bioinformatics analyses of the biologic processes and signaling pathways that were changed in the BD group and constructed a proteomics landscape of organ-resolved BD pathogenesis. We then validated the biomarkers of disease severity and the vascular subset in an independent cohort of 108 BD patients and 29 healthy controls using an enzyme-linked immunosorbent assay. RESULTS: The BD group had 220 differentially expressed proteins, which discriminated between BD patients (88.6%) and healthy controls (95.5%). The bioinformatics analyses revealed different biologic processes associated with BD pathogeneses, including complement activation, wound healing, angiogenesis, and leukocyte-mediated immunity. Furthermore, the constructed proteomics landscape of organ-resolved BD identified proteomics features of BD associated with different organs and protein targets that could be used for the development of therapeutic treatment. Hyaluronic binding protein 2, tenascin, and serpin A3 were validated as potential biomarkers for the clinical assessment of vascular BD and treatment targets. CONCLUSION: Our results provide valuable insight into the pathogenesis of organ-resolved BD in terms of proteomics characteristics and potential biomarkers for clinical assessment and potential therapies for vascular BD.

Development of a protein microarray for profiling circulating autoantibodies in human diseases.

PURPOSE: To develop a robust microarray platform to detect thousands of serological autoantibodies (AAbs) simultaneously in different diseases. EXPERIMENTAL DESIGN: An AAbMap microarray was prepared by printing a total of 4032 purified His-tagged human proteins and peptide probes on a chemically-modified slide. The sensitivity, dynamic range, and the inter- and intra-array reproducibility of the AAb microarray were then systematically tested and optimized. Finally, the large-scale profiling of AAbs in the serum of patients with different human diseases using the AAbMap microarray was demonstrated. RESULTS: The dynamic range of antibody (Ab) detection was 2 to 3 orders of magnitude with the lowest limit of detection (LOD) of 68 pg/mL. The intra-array (r) correlation of duplicate spots was 1.00, whereas the inter-array correlations between different arrays and batches were 0.99 and 0.97 to 0.98, respectively. Notably, 132, 266, 171, and 84 AAbs were detected in pooled serum from healthy controls (HCs) or patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or lung cancer (LC), respectively. These AAbs included antibodies that target well-known disease biomarkers, such as anti-cyclic citrullinated peptide, anti-ribonucleoprotein, and anti-nucleosome. CONCLUSIONS AND CLINICAL RELEVANCE: We developed a microarray platform to measure thousands of serological AAbs simultaneously with high sensitivity and reproducibility. The array can help study autoimmunity and complement genomics, proteomics, and metabolomics data for systematic investigations of human diseases.

Inhibitor screening using microarray identifies the high capacity of neutralizing antibodies to Spike variants in SARS-CoV-2 infection and vaccination.

Rationale: Mutations of SARS-CoV-2, which is responsible for coronavirus disease 2019 (COVID-19), could impede drug development and reduce the efficacy of COVID-19 vaccines. Here, we developed a multiplexed Spike-ACE2 Inhibitor Screening (mSAIS) assay that can measure the neutralizing effect of antibodies across numerous variants of the coronavirus's Spike (S) protein simultaneously. Methods: The SARS-CoV-2 spike variant protein microarrays were prepared by printing 72 S variants onto a chemically-modified glass slides. The neutralization potential of purified anti-S antibodies and serum from convalescent COVID-19 patients and vaccinees to S variants were assessed with the mSAIS assay. Results: We identified new S mutations that are sensitive and resistant to neutralization. Serum from both infected and vaccinated groups with a high titer of neutralizing antibodies (NAbs) displayed a broader capacity to neutralize S variants than serum with low titer NAbs. These data were validated using serum from a large vaccinated cohort (n = 104) with a tiled S peptide microarray. In addition, similar results were obtained using a SARS-CoV-2 pseudovirus neutralization assay specific for wild-type S and five prevalent S variants (D614G, B.1.1.7, B.1.351, P.1, B.1.617.2), thus demonstrating that high antibody diversity is associated with high NAb titers. Conclusions: Our results demonstrate the utility of the mSAIS platform in screening NAbs. Moreover, we show that heterogeneous antibody populations provide a more protective effect against S variants, which may help direct COVID-19 vaccine and drug development.