Rapid visualization of PD-L1 expression level in glioblastoma immune microenvironment via machine learning cascade-based Raman histopathology.

INTRODUCTION: Combination immunotherapy holds promise for improving survival in responsive glioblastoma (GBM) patients. Programmed death-ligand 1 (PD-L1) expression in immune microenvironment (IME) is the most important predictive biomarker for immunotherapy. Due to the heterogeneous distribution of PD-L1, post-operative histopathology fails to accurately capture its expression in residual tumors, making intra-operative diagnosis crucial for GBM treatment strategies. However, the current methods for evaluating the expression of PD-L1 are still time-consuming. OBJECTIVE: To overcome the PD-L1 heterogeneity and enable rapid, accurate, and label-free imaging of PD-L1 expression level in GBM IME at the tissue level. METHODS: We proposed a novel intra-operative diagnostic method, Machine Learning Cascade (MLC)-based Raman histopathology, which uses a coordinate localization system (CLS), hierarchical clustering analysis (HCA), support vector machine (SVM), and similarity analysis (SA). This method enables visualization of PD-L1 expression in glioma cells, CD8+ T cells, macrophages, and normal cells in addition to the tumor/normal boundary. The study quantified PD-L1 expression levels using the tumor proportion, combined positive, and cellular composition scores (TPS, CPS, and CCS, respectively) based on Raman data. Furthermore, the association between Raman spectral features and biomolecules was examined biochemically. RESULTS: The entire process from signal collection to visualization could be completed within 30 min. In an orthotopic glioma mouse model, the MLC-based Raman histopathology demonstrated a high average accuracy (0.990) for identifying different cells and exhibited strong concordance with multiplex immunofluorescence (84.31 %) and traditional pathologists' scoring (R2 ≥ 0.9). Moreover, the peak intensities at 837 and 874 cm-1 showed a positive linear correlation with PD-L1 expression level. CONCLUSIONS: This study introduced a new and extendable diagnostic method to achieve rapid and accurate visualization of PD-L1 expression in GBM IMB at the tissular level, leading to great potential in GBM intraoperative diagnosis for guiding surgery and post-operative immunotherapy.

Tumor identification via in vivo portable Raman detection of sialic acid with a dual gold nanoprobe system.

A dual gold nanoprobe system was designed for in vivo portable Raman detection of sialic acid (SA) for tumor identification. The dual gold nanoprobe system contained two gold nanoprobes, Au10-DTTC/PEG-PBA and Au40-PEG-SA. Au10-DTTC/PEG-PBA was constructed on a 10 nm gold nanoparticle modified with 3,3'-diethylthia tricarbocyanine iodide (DTTCI) as the Raman reporter and 3-aminophenylboronic acid (APBA) through a thiol PEG succinimidyl carboxymethyl ester (HS-PEG-NHS) linker for specific recognition of SA. Au40-PEG-SA was constructed on a 40 nm gold nanoparticle modified with SA through HS-PEG-NHS. For in vivo detection of SA, Au10-DTTC/PEG-PBA and Au40-PEG-SA were subsequently injected into tumor xenografted mice with optimal interval and retention times. Through the specific recognition between PBA and SA, the conjugates of Au10-DTTC/PEG-PBA and Au40-PEG-SA formed in the tumor region emitted strong SERS signals of DTTC, which could be detected by a portable Raman detector. This work provides a convenient and portable method to detect SA in tumor xenografted mice, which is useful for family-stay identification and clinical cleavage of tumors.

O-GlcNAcylation mapping of single living cells by in situ quantitative SERS imaging.

O-GlcNAcylation is involved in many biological processes including cancerization. Nevertheless, its in situ quantification in single living cells is still a bottleneck. Here we develop a quantitative SERS imaging strategy for mapping the O-GlcNAcylation distribution of single living cells. O-GlcNAcylated compounds (OGCs) can be quantified through their in situ azide labeling and then a click reaction competing with azide and Raman reporter labeled 15 nm-gold nanoparticles (AuNPs) for linking to dibenzocyclooctyne labeled 40 nm-AuNPs to produce OGC-negatively correlated SERS signals. The calibration curve obtained in vitro can be conveniently used for detecting OGCs in different areas of single living cells due to the negligible effect of cell medium on the click linkage and Raman signal. This method has been successfully applied in mapping O-GlcNAcylation distribution in different cell lines and monitoring O-GlcNAcylation variation during cell cycling, which demonstrate its great practicability and expansibility in glycosylation related analysis.

Gold Nanostar@Polyaniline Theranostic Agent with High Photothermal Conversion Efficiency for Photoacoustic Imaging-Guided Anticancer Phototherapy at a Low Dosage.

Due to the strong and tunable photothermal effect, metallic nanoparticles are of enormous interest in light-activated biomedical applications, such as photoacoustic imaging (PAI) and photothermal therapy (PTT). However, the photothermal conversion efficiency (PCE) of existing metallic photothermal agents is still unsatisfactory. Herein, we develop an efficient photothermal theranostic agent based on a gold nanostar@polyaniline core-shell nanocomposite with high PCE for PAI-guided PTT at a low dosage. After optimizing the relative composition of polyaniline (PANI) and gold nanostars (AuNSs), this nanocomposite eventually empowers an outstanding PCE of up to 78.6%, which is much better than AuNSs or PANI alone and most of the existing photothermal theranostic agents. Besides, the nanocomposite can act as a targeted probe for tumors by hyaluronic acid (HA) modification without compromising the photothermal performance. The obtained nanoprobes named AuNSPHs exhibit promising biocompatibility and great performance of PAI-guided PTT to treat triple-negative breast cancer both in vitro and in vivo. More importantly, a single injection of AuNSPHs significantly suppresses tumor growth with a low dosage of Au (0.095 mg/kg), which is attributed to the high PCE of AuNSPHs. Taking advantage of the exhilarating photothermal conversion ability, this theranostic agent can safely potentiate the antitumor therapeutic efficacy of laser-induced ablation and holds great potential for future medical applications.

A pore-forming protein-induced surface-enhanced Raman spectroscopic strategy for dynamic tracing of cell membrane repair.

The plasma membrane repair holds significance for maintaining cell survival and homeostasis. To achieve the sensitive visualization of membrane repair process for revealing its mechanism, this work designs a perforation-induced surface-enhanced Raman spectroscopy (SERS) strategy by conjugating Raman reporter (4-mercaptobenzoic acid) loaded gold nanostars with pore-forming protein streptolysin O (SLO) to induce the SERS signal on living cells. The SERS signal obviously decreases with the initiation of membrane repair and the degradation of SLO pores due to the departure of gold-nanostar-conjugated SLO. Thus, the designed strategy can dynamically visualize the complete cell membrane repair and provide a sensitive method to demonstrate the SLO endocytosis- and exocytosis-mediated repairing mechanism. Using DOX-resistant MCF-7 cells as a model, a timely repair-blocking technology for promoting the highly efficient treatment of drug-resistant cancer cells is also proposed. This work opens an avenue for probing the plasma membrane repairing mechanisms and designing the precision therapeutic schedule.

A Filter Supported Surface-Enhanced Raman Scattering "Nose" for Point-of-Care Monitoring of Gaseous Metabolites of Bacteria.

This work designs a convenient method for fabrication of surface-enhanced Raman scattering (SERS) devices by loading gold nanostars (AuNSs) on a flat filter support with vacuum filtration. The dense accumulation of AuNSs results in a strong sensitization to SERS signal and shows sensitive response to gaseous metabolites of bacteria, which produces a SERS "nose" for rapid point-of-care monitoring of these metabolites. The "nose" shows good reproducibility and stability and can be used for SERS quantitation of a gaseous target with Raman signal. The impressive performance of the proposed SERS "nose" for detecting gaseous metabolites of common foodborne bacteria like Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa from inoculated samples demonstrates its much higher sensitivity than that of human sense and application in distinguishing spoiled food at an early stage and real-time tracing of food spoilage degree. The strong point-of-care testing ability of the designed SERS "nose" and the miniaturization of whole equipment extend greatly the analytical application of SERS technology in food safety and public health.

"Three-in-One" SERS Adhesive Tape for Rapid Sampling, Release, and Detection of Wound Infectious Pathogens.

The traditional colony culture method for detection of pathogens is subjected to the laborious and tedious experimental procedure, which limits its application in point-of-care (POC) testing and quick diagnosis. This work designs an intelligent adhesive tape as a "three-in-one" platform for rapid sampling, photocontrolled release, and surface-enhanced Raman scattering (SERS) detection of pathogens from infected wounds. This tape is constructed by encapsulating densely packed gold nanostars as SERS substrates between two pieces of graphene and modified with a synthetic o-nitrobenzyl derivative molecule to form an artificial biointerface for highly efficient pathogen capture via electrostatic interaction. The captured targets can be conveniently released onto a solid culture medium by UV cleavage of o-nitrobenzyl moiety for pathogen growth and in situ SERS detection. As a proof of strategy, this "three-in-one" platform has been used for detecting the concurrent infection of Pseudomonas aeruginosa and Staphylococcus aureus by pasting the tape on a skin burn wound. The impressive detection performance with an analytical time of only several hours for these pathogens at an early growth stage demonstrates its great potential as a POC testing device for health care.

A multifunctional SERS sticky note for real-time quorum sensing tracing and inactivation of bacterial biofilms.

Quorum sensing (QS) is crucial for bacterial survival and activity. Although detecting related signaling metabolites can reveal QS, a versatile platform for convenient real-time imaging of their secretion in the context of bacterial biofilms along with inhibition to the growth of biofilms is still highly desired. Here we develop a flexible sticky note with a sandwich structure by encapsulating gold nanostars between two pieces of hexagonal boron nitride layers, which can be easily pasted on natural biofilms to monitor in real-time the secreted signaling molecule by SERS imaging with high sensitivity and spatiotemporal resolution. Using Pseudomonas aeruginosa and its pyocyanin secretion as a model and an internal standard for self-calibration of SERS signals, the sticky note achieves reliable quantification and a rapid response to the secretion as early as 1 h biofilm growth. With antibiotic loading, the multifunctional SERS sticky note also demonstrated effective inactivation of the bacterial biofilm with simultaneous evaluation of the inactivation effect. This multifunctional SERS sticky note provides a versatile platform for investigating bacterial behaviors and developing antibacterial therapeutics.

Development of "Purple Endosperm Rice" by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System.

Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice (Oryza sativa L.), could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes (two regulatory genes from maize and six structural genes from Coleus) driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm "Purple Endosperm Rice" (called "Zijingmi" in Chinese), which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation (or enhancement) of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.

Polyadenine-Modulated DNA Conformation Monitored by Surface-Enhanced Raman Scattering (SERS) on Multibranched Gold Nanoparticles and Its Sensing Application.

This work proposes a facile way to modulate the conformation of DNA from the "Lie-Down" to the "Stand-Up" conformation on the surface of multibranched gold nanoparticles (AuNPs). This is realized by regulating the length of polyadenine (polyA) linked to the DNA sequence and/or the hybridization of this sequence with the target DNA, and can be monitored by the Raman signal owing to the excellent performance of multibranched AuNPs (AuNSs) as a surface-enhanced Raman scattering (SERS) substrate and the distance change between the Raman reporter and the substrate. The probable mechanism, which depends on the repulsion of polyA from the sequence and the tip assembly, has also been probed through theoretical simulation using the finite difference time domain method. By virtue of this strategy, a conformation-transformation-based DNA@AuNS sensor is constructed for the identification of a specific oligonucleotide, which has been used for the detection of DNA sequences associated with Severe Acute Respiratory Syndrome (SARS). This strategy leads to a novel sensing platform with good extendibility for DNA analysis, and provides a powerful protocol for facilitating the cognition of DNA conformation on metal surfaces.

A three-dimensional nitrogen-doped graphene structure: a highly efficient carrier of enzymes for biosensors.

In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme-based biosensors. Owing to the high conductivity, large porosity and tunable nitrogen-doping ratio, this kind of graphene framework shows outstanding electrical properties and a large surface area for enzyme loading and biocatalytic reactions. Using glucose oxidase (GOx) as a model enzyme and chitosan (CS) as an efficient molecular binder of the enzyme, our 3D-NG based biosensors show extremely high sensitivity for the sensing of glucose (226.24 µA mM(-1) m(-2)), which is almost an order of magnitude higher than those reported in most of the previous studies. The stable adsorption and outstanding direct electrochemical behaviour of the enzyme on the nanocomposite indicate the promising application of this 3D enzyme carrier in high-performance electrochemical biosensors or biofuel cells.