Single-Cell Spatial MIST for Versatile, Scalable Detection of Protein Markers.

High-multiplex detection of protein biomarkers across tissue regions has been an attractive spatial biology approach due to significant advantages over traditional immunohistochemistry (IHC) methods. Different from most methods, spatial multiplex in situ tagging (MIST) transfers the spatial protein expression information to an ultrahigh-density, large-scale MIST array. This technique has been optimized to reach single-cell resolution by adoption of smaller array units and 30% 8-arm PEG polymer as transfer medium. Tissue cell nuclei stained with lamin B have been clearly visualized on the MIST arrays and are colocalized with detection of nine mouse brain markers. Pseudocells defined at 10 µm in size have been used to fully profile tissue regions including cells and the intercellular space. We showcased the versatility of our technology by successfully detecting 20 marker proteins in kidney samples with the addition of five minutes atop the duration of standard immunohistochemistry protocols. Spatial MIST is amenable to iterative staining and detection on the same tissue samples. When 25 proteins were co-detected on 1 mouse brain section for each round and 5 rounds were executed, an ultrahigh multiplexity of 125 proteins was obtained for each pseudocell. With its unique abilities, this single-cell spatial MIST technology has the potential to become an important method in advanced diagnosis of complex diseases.

Magnetic Nanoparticles for Protein Separation and Purification.

Proteins are essential for various functions such as brain activity and muscle contraction in humans. Even though food is a source of proteins, the bioavailability of proteins in most foods is usually limited due to matrix interaction with other biomolecules. Thus, it is essential to extract these proteins and provide them as a nutraceutical supplement to maintain protein levels and avoid protein deficiency. Hence, protein purification and extraction from natural sources are highly significant in biomedical applications. Chromatography, crude mechanical disruption, use of extractive chemicals, and electrophoresis are some of the methods applied to isolate specific proteins. Even though these methods possess several advantages, they are unable to extract specific proteins with high purity. A suitable alternative is the use of nanoparticles, which can be beneficial in protein purification and extraction. Notably, magnetic iron and iron-based nanoparticles have been employed in protein extraction processes and can be reused via demagnetization due to their magnetic property, smaller size, morphology, high surface-to-volume ratio, and surface charge-mediated property. This chapter is a summary of various magnetic nanoparticles (MNPs) that can be used for the biomolecular separation of proteins.

Plasmonic Alloys Reveal a Distinct Metabolic Phenotype of Early Gastric Cancer.

Gastric cancer (GC) is a multifactorial process, accompanied by alterations in metabolic pathways. Non-invasive metabolic profiling facilitates GC diagnosis at early stage leading to an improved prognostic outcome. Herein, mesoporous PdPtAu alloys are designed to characterize the metabolic profiles in human blood. The elemental composition is optimized with heterogeneous surface plasmonic resonance, offering preferred charge transfer for photoinduced desorption/ionization and enhanced photothermal conversion for thermally driven desorption. The surface structure of PdPtAu is further tuned with controlled mesopores, accommodating metabolites only, rather than large interfering compounds. Consequently, the optimized PdPtAu alloy yields direct metabolic fingerprints by laser desorption/ionization mass spectrometry in seconds, consuming 500 nL of native plasma. A distinct metabolic phenotype is revealed for early GC by sparse learning, resulting in precise GC diagnosis with an area under the curve of 0.942. It is envisioned that the plasmonic alloy will open up a new era of minimally invasive blood analysis to improve the surveillance of cancer patients in the clinical setting.

Shrimp shells extracted chitin in silver nanoparticle synthesis: Expanding its prophecy towards anticancer activity in human hepatocellular carcinoma HepG2 cells.

In this study, a well-organized, simplistic, and biological route of AgNPs (AgNPs) was synthesized using shrimp shell extracted chitin as reducing, capping and stabilizing factor under the optimized conditions. Also, the anticancer potential of synthesized biogenic AgNPs was evaluated against human hepatocarcinoma (HepG2) cells. Ultraviolet visible spectroscopy (UV-Vis spec) study indicated that the development of AgNPs present in the colloidal solution was single peak at 446 nm. FTIR results showed a strong chemical interaction between the chitin and biogenic AgNPs; whereas, XRD studies confirmed AgNPs presence in the composites. The SEM TEM analytical studies confirmed the synthesized AgNPs had a spherical shape crystalline structure with size ranges from 17 to 49 nm; EDX study also confirmed the percentage of weight and atomic elements available in the colloidal mixture. Furthermore, the synthesized AgNPs showed significant cytotoxic effect on the HepG2 cells with an IC50 value shown at 57 ± 1.5 µg/ml. The apoptotic and necrotic cell death effects of AgNPs were also confirmed by flow cytometry. The upregulated apoptotic related proteins Bax, cytochrome-c, caspase-3, caspase-9, PARP and downregulated anti-apoptotic related proteins Bcl-2 and Bcl-xl in cancer cells, confirmed the anticancer potential of AgNPs. These findings suggest that the AgNPs possess significant anticancer activity against HepG2 cells which could play major role in the therapeutic drug development to treat cancer in future.

Zirconia Hybrid Nanoshells for Nutrient and Toxin Detection.

Monitoring milk quality is of fundamental importance in food industry, because of the nutritional value and resulting position of milk in daily diet. The detection of small nutrients and toxins in milk is challenging, considering high sample complexity and low analyte abundance. In addition, the slow analysis and tedious sample preparation hinder the large-scale application of conventional detection techniques. Herein, zirconia hybrid nanoshells are constructed to enhance the performance of laser desorption/ionization mass spectrometry (LDI MS). Zirconia nanoshells with the optimized structures and compositions are used as matrices in LDI MS and achieve direct analysis of small molecules from 5 nL of native milk in ≈1 min, without any purification or separation. Accurate quantitation of small nutrient is achieved by introducing isotope into the zirconia nanoshell-assisted LDI MS as the internal standard, offering good consistency to biochemical analysis (BCA) with R2  = 0.94. Further, trace toxin is enriched and identified with limit-of-detection (LOD) down to 4 pm, outperforming the current analytical methods. This work sheds light on the personalized design of material-based tool for real-case bioanalysis and opens up new opportunities for the simple, fast, and cost-effective detection of various small molecules in a broad field.

Machine learning of serum metabolic patterns encodes early-stage lung adenocarcinoma.

Early cancer detection greatly increases the chances for successful treatment, but available diagnostics for some tumours, including lung adenocarcinoma (LA), are limited. An ideal early-stage diagnosis of LA for large-scale clinical use must address quick detection, low invasiveness, and high performance. Here, we conduct machine learning of serum metabolic patterns to detect early-stage LA. We extract direct metabolic patterns by the optimized ferric particle-assisted laser desorption/ionization mass spectrometry within 1 s using only 50 nL of serum. We define a metabolic range of 100-400 Da with 143 m/z features. We diagnose early-stage LA with sensitivity~70-90% and specificity~90-93% through the sparse regression machine learning of patterns. We identify a biomarker panel of seven metabolites and relevant pathways to distinguish early-stage LA from controls (p < 0.05). Our approach advances the design of metabolic analysis for early cancer detection and holds promise as an efficient test for low-cost rollout to clinics.

Detection and Inhibition of Bacteria on a Dual-Functional Silver Platform.

Detection and inhibition of bacteria are universally required in clinics and daily life for health care. Developing a dual-functional material is challenging and in demand, engaging advanced applications for both defined bioanalysis and targeted biotoxicity. Herein, magnetic silver nanoshells are designed as a multifunctional platform for the detection and inhibition of bacteria. The optimized magnetic silver nanoshells enable direct laser desorption/ionization mass spectrometry based metabolic analysis of bacteria (≈10 µL-1 ), in complex biofluids. The serum infection process (0-10 h) is monitored by statistics toward clinical classification. Moreover, magnetic silver nanoshells facilitate surface adhesion on bacteria due to nanoscale surface roughness and thus display long-term antibacterial effects. Bacteria metabolism is studied with metabolic biomarkers (e.g., malate and lysine) identified during inhibition, showing cell membrane destruction and dysfunctional protein synthesis mechanisms. This work not only guides the design of material-based approaches for bioanalysis and biotoxicity, but contributes to bacteria-related diagnosis by using specific metabolic biomarkers for sensitive detection and new insights by monitoring metabolomic change of bacteria for antibacterial applications.

Metabolic Fingerprinting on a Plasmonic Gold Chip for Mass Spectrometry Based in Vitro Diagnostics.

Current metabolic analysis is far from ideal to engage clinics and needs rationally designed materials and device. Here we developed a novel plasmonic chip for clinical metabolic fingerprinting. We first constructed a series of chips with gold nanoshells on the surface through controlled particle synthesis, dip-coating, and gold sputtering for mass production. We integrated the optimized chip with microarrays for laboratory automation and micro-/nanoscaled experiments, which afforded direct high-performance metabolic fingerprinting by laser desorption/ionization mass spectrometry using 500 nL of various biofluids and exosomes. Further we for the first time demonstrated on-chip in vitro metabolic diagnosis of early stage lung cancer patients using serum and exosomes. This work initiates a new bionanotechnology based platform for advanced metabolic analysis toward large-scale diagnostic use.

Plasmonic silver nanoshells for drug and metabolite detection.

In-vitro metabolite and drug detection rely on designed materials-based analytical platforms, which are universally used in biomedical research and clinical practice. However, metabolic analysis in bio-samples needs tedious sample preparation, due to the sample complexity and low molecular abundance. A further challenge is to construct diagnostic tools. Herein, we developed a platform using silver nanoshells. We synthesized SiO2@Ag with tunable shell structures by multi-cycled silver mirror reactions. Optimized nanoshells achieved direct laser desorption/ionization mass spectrometry in 0.5 µL of bio-fluids. We applied these nanoshells for disease diagnosis and therapeutic evaluation. We identified patients with postoperative brain infection through daily monitoring and glucose quantitation in cerebrospinal fluid. We measured drug distribution in blood and cerebrospinal fluid systems and validated the function of blood-brain/cerebrospinal fluid-barriers for pharmacokinetics. Our work sheds light on the design of materials for advanced metabolic analysis and precision diagnostics.Preparation of samples for diagnosis can affect the detection of biomarkers and metabolites. Here, the authors use a silver nanoparticle plasmonics approach for the detection of biomarkers in patients as well as investigate the distribution of drugs in serum and cerebral spinal fluid.

Antitumor Effect of the Mannich Base(1,3-bis-((3-Hydroxynaphthalen-2-yl)phenylmethyl)urea) on Hepatocellular Carcinoma.

The present study was designed to evaluate the antitumor effects of the synthetic Mannich base 1,3-bis-((3-hydroxynaphthalen-2-yl)phenylmethyl)urea (1,3-BPMU) against HEP-G2 hepatoma cells and diethylnitrosamine (DEN)-induced hepatocarcinoma (HCC) in albino rats. In vitro analysis results revealed that 1,3-BPMU showed significant cytotoxicity and cell growth inhibition in HEP-G2 hepatoma cells in a concentration-dependent manner. Furthermore, flow cytometry results indicated that 1,3-BPMU enhanced early and late apoptosis. The maximum apoptosis was exhibited at a concentration of 100 µg/mL of 1,3-BPMU. In in vivo analysis, DEN treatment increased the content of nucleic acids, LPO and the activities of AST, ALT, ALP, LDH, γGT and 5'NT with decreased antioxidant activity as compared to control rats. However, 1,3-BPMU treatment to DEN-induced rats decreased the content of nucleic acids, LPO and the activities of AST, ALT, ALP, LDH, γGT and 5'NT and increased the activities of SOD, CAT, GPx, GST and GR (p < 0.05). Furthermore, 1,3-BPMU enhanced the apoptosis via upregulation of caspase-3 and caspase-9 and the downregulation of Bcl-2 and Bcl-XL mRNA expression as compared to DEN-induced rats. Histological and ultrastructural investigation showed that 1,3-BPMU treatment renovated the internal architecture of the liver in DEN-induced rats. In this study, the molecular and pre-clinical results obtained by treatment of DEN-induced rats with 1,3-BPMU suggested that 1,3-BPMU might be considered as an antitumor compound in the future.

Identification of Differential Protein Expression in Hepatocellular Carcinoma Induced Wistar Albino Rats by 2D Electrophoresis and MALDI-TOF-MS Analysis.

Hepato cellular carcinoma (HCC) is a type of malignant tumor. To investigate the proteins in cancer molecular mechanism and its role in HCC, we have used proteomic tools such as 2DE and MALDI-TOF-MS. Our investigation ravels that, plasma α-fetoprotein and carcinoembryonic antigen levels were elevated in DEN induced rats and gradually decreased after the treatment with 1,3BPMU. 2DE and MALDI-TOF-MS tool offers to identify the up and down regulation of proteins in HCC. Proteomic study reveals that, five differentially expressed proteins were identified in DEN induced rats and 1,3BPMU treated rats i.e. three up regulated protein such as T kininogen, NDPKB, PRMT1 (DEN induced rats), RGS19 and PAF (1,3BPMU treated rats) in 3BPMU treated rats, activation of transcription of a single gene from multiple promoters provides flexibility in the controlled gene expression. The regulations of hepatocyte stimulating factor were slow down the proliferation of hepatic cell and uncontrolled hepatic cell growth and also molecular signals strongly argue for a patho-physiological role in liver metastasis to control the cell aggression. This indicates that, anti cancer property of 1,3BPMU can be used as potent anti cancer agent. The present study also shows the proteomic approach helps to elucidate the tumor maker as well as regulatory marker proteins in HCC.