Identification of Molecular Profile of Cell Membrane via Magnetic Plasmonic Nanoprobe.

Cell membranes are primarily composed of lipids, membrane proteins, and carbohydrates, and the related studies of membrane components and structures at different stages of disease development, especially membrane proteins, are of great significance. Here, we investigate the chemical signature profiles of cell membranes as biomarkers for cancer cells via label-free surface-enhanced Raman scattering (SERS). A magnetic plasmonic nanoprobe was proposed by decorating magnetic beads with silver nanoparticles, allowing self-driven cell membrane-targeted accumulation within 5 min. SERS profiles of three types of breast cells were achieved under the plasmon enhancement effect of these nanoprobes. Membrane fingerprint spectra from breast cell lines were further classified with the convolutional neural network model, which perfectly distinguished between two breast cancer subtypes. We further tested various clinical samples using this method and obtained fingerprint spectra from primary cells and frozen slices. This study presents a practical, user-friendly approach for label-free and in situ analysis of cell membranes, which can work for early tumor screening and treatment assessment for tumors reliant on the Molecular profiles of cell membranes. Additionally, this method can be applied universally to explore cell membrane components of other cells, thus assisting Human Cell Atlas.

Fabrication of High-Negatively Charged Bicelle-Mediated Supported Lipid Bilayer.

Supported lipid bilayers (SLBs), two-dimensional lipid films formed on a solid-supporting substrate, serve as models for biomembranes and exhibit remarkable potential in chemistry, biology, and medicine. However, preparing SLBs with highly negatively charged contents on the negatively charged surface by overcoming electrostatic repulsion remains a challenge. Here, a creative bicelle-mediated and divalent cation-free SLB preparation method with the assistance of phosphate-buffered saline (PBS) solution was proposed, which can form the SLBs containing 50% DOPS or 30% CL on the silica surface monitored by a quartz crystal microbalance with dissipation (QCM-D). Results of molecular dynamics (MD) simulation indicate that electrostatic repulsion can be overcome by the increased number of hydrogen bonds caused by the adsorption of dihydrogen phosphate ions onto the headgroups of lipids. In addition, the negatively charged SLB formation was identified to be a three-step kinetic process, which differs from a two-step mechanism in the case of amphoteric SLB. The extra kinetic step can be attributed to the reduction in the number of intermolecular hydrogen bonds and the ordering of water molecules in the hydration layer. This investigation resolves the challenge of fabricating SLB over negatively charged surfaces and offers a fresh perspective on the SLB assembly methodology.

Sensitive detection of genetically modified maize based on a CRISPR/Cas12a system.

With the vigorous development of biotechnology, genetically modified organisms (GMOs) have become more and more common. In order to effectively supervise and administrate them, the rapid and accurate detection of GMOs is urgently demanded. Here, GMO gene-specific sensing methods based on colorimetry and surface-enhanced Raman scattering (SERS) were proposed based on the lateral branch cleavage function of the CRISPR/Cas12a system. Two transgenes, pCaMV35S and M810 Cry1Ab, were chosen as targets for transgenic crops. By using these methods, we performed transgenic detection on five types of maize leaves and successfully distinguished transgenic from non-transgenic samples. The colorimetric method is rapid, economical and available for field detection. The SERS approach, giving a higher sensitivity to 100 fM, is more suitable for laboratory application scenarios. This study explores practical transgenic detection approaches and will be valuable for the supervision of GMOs.

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

Single-Cell Analysis and Classification according to Multiplexed Proteins via Microdroplet-Based Self-Driven Magnetic Surface-Enhanced Raman Spectroscopy Platforms Assisted with Machine Learning Algorithms.

A microdroplet-based surface-enhanced Raman spectroscopy (microdroplet SERS) platform was constructed to envelop individual cells in microdroplets, followed by the SERS detection of their extracellular vesicle-proteins (EV-proteins) via the in-drop immunoassays by use of immunomagnetic beads (iMBs) and immuno-SERS tags (iSERS tags). A unique phenomenon is found that iMBs can start a spontaneous reorientation on the probed cell surface based on the electrostatic force-driven interfacial aggregation effect, which leads EV-proteins and iSERS tags to be gathered from a liquid phase to a cell membrane interface and significantly improves SERS sensitivity to the single-cell analysis level due to the formation of numbers of SERS hotspots. Three EV-proteins from two breast cancer cell lines were collected and further analyzed by machine learning algorithmic tools, which will be helpful for a deeper understanding of breast cancer subtypes from the view of EV-proteins.

Asymmetric Heterodimer-Regulated Surface Plasmon Coupling ECL Polarization Strategy for MiRNA-182 Detection.

In this work, a novel plasmonic heterodimer with controllable hot spot was designed and applied to regulate surface plasmon coupling electrochemiluminescence (SPC-ECL) polarization sensing system. The heterodimer nanostructure consisted of individual Au-Ag core-shell nanocubes (Au@Ag NC) and Au nanospheres (Au NS), which were precisely assembled by thiol-DNA and biotin-streptavidin. The asymmetric nanostructure can significantly modulate the ECL intensity and emission polarization angle based on the synergy of the surface plasmon coupling (SPC) effect and the lightning rod effect with extraordinary field enhancement in the hot spot region. As a result, the isotropic ECL signal of zinc-doped nitrogen dots (Zn-N dots) was regulated in the directional emission. Furthermore, the SPC-ECL biosensor was successfully applied to detect miRNA-182 in triple-negative breast cancer (TNBC) tissues. The research on the established relationship between ECL polarization analysis and plasmonic heterodimers can provide a new pathway for the development of ECL sensing platforms.

Direct Virus Gene Detection: A CRISPR/dCas9-Mediated Surface-Enhanced Raman Scattering Strategy with Enzyme-Catalyzed Signal Amplification.

The aberrant growth of cervical cells caused by the infection of human papillomavirus (HPV) may cause cervical cancer. In order to effectively prevent the occurrence of cervical cancer and for better follow-up treatment after surgery, a rapid and reliable detection method of HPV DNA is essential. Here, a surface-enhanced Raman scattering (SERS) detection method was developed based on the clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 technique and the enzyme catalysis amplification reaction, which achieved a simple and rapid detection of low-content HPV genes. The CRISPR/dCas9/sgRNA complex was anchored above a magnetic bead, which can precisely capture target DNA sequences, exhibiting high selectivity for HPV genes. When the biotinylated target DNAs exist, they can bridge a streptavidin-modified horse radish peroxidase (HRP) to the magnetic bead, producing an HRP-decorated conjugate. This conjugate allows an HRP-catalyzed reaction for its substrate (3,3',5,5'-tetramethylbenzidine, TMB). Gold nanostars with a silica shell exhibiting the lightning rod effect of SERS were employed to measure the SERS spectra of the oxidative product of TMB. Enzyme catalysis and SERS co-contribute to the SERS signal output, ensuring a high detection sensitivity. This method is a proof of concept for detecting HPV DNAs in a complex system. The current method can be applied to other target DNAs simply by changing the sgRNA sequence. Many superiors portend that the CRISPR/dCas9-based SERS method is promising for further clinical application.

A SERS/fluorescence dual-mode immuno-nanoprobe for investigating two anti-diabetic drugs on EGFR expressions.

A surface-enhanced Raman scattering (SERS)/fluorescence dual-mode nanoprobe was proposed to assess anti-diabetic drug actions from the expression level of the epidermal growth factor receptor (EGFR), which is a significant biomarker of breast cancers. The nanoprobe has a raspberry shape, prepared by coating a dye-doped silica nanosphere with a mass of SERS tags, which gives high gains in fluorescence imaging and SERS measurement. The in situ detection of EGFR on the cell membrane surfaces after drug actions was achieved by using this nanoprobe, and the detection results agree with the enzyme-linked immunosorbent assay (ELISA) kit. Our study suggests that rosiglitazone hydrochloride (RH) may be a potential drug for diabetic patients with breast cancer, while the anti-cancer effect of metformin hydrochloride (MH) is debatable since MH slightly promotes the EGFR expression of MCF-7 cells in this study. This sensing platform endows more feasibility for highly sensitive and accurate feedback of pesticide effects at the membrane protein level.

Gold Nanorod Vertical Array-Based Electrochemiluminescence Polarization Assay for Triple-Negative Breast Cancer Detection.

In this work, a polarization-resolved electrochemiluminescence (ECL) sensor for microRNA-155 (miRNA-155) detection has been constructed based on the surface plasmon coupling effect. In the sensing system, nitrogen dots (N dots) were employed as ECL emitters. As a surface-enhanced structure, a gold nanorod vertical array was constructed on the electrode surface by the volatilization-induced self-assembly. The coupling of the adjacent gold nanorods in the array can generate significant local electromagnetic fields. Due to the anisotropy of gold nanorods and the hot spot effect of the vertical array, the ECL signal of N dots was greatly improved at a specific polarization angle. In addition, the catalytic hairpin self-assembly strategy was used to amplify the nucleic acid analyte signal. As a result, the polarization-resolved ECL sensor can detect miRNA-155 sensitively, which is related to triple-negative breast cancer.

Single-Cell VEGF Analysis by Fluorescence Imaging-Microfluidic Droplet Platform: An Immunosandwich Strategy on the Cell Surface.

Despite recent advances in single-cell analysis techniques, the ability of single-cell analysis platforms to track specific cells that secreted cytokines remains limited. Here, we report a microfluidic droplet-based fluorescence imaging platform that can analyze single cell-secreted vascular endothelial growth factor (VEGF), an important regulator of physiological and pathological angiogenesis, to explore cellular physiological clues at the single-cell level. Two kinds of silica nanoparticle (NP)-based immunoprobes were developed, and they were bioconjugated to the membrane proteins of the probed cell surface via the bridging of secreted VEGF. Thus, an immunosandwich assay was built above the probed cell via fluorescence imaging analysis of each cell in isolated droplets. This analytical platform was used to compare the single-cell VEGF secretion ability of three cell lines (MCF-7, HeLa, and H8), which experimentally demonstrates the cellular heterogeneity of cells in secreting cytokines. The uniqueness of this method is that the single-cell assay is carried out above the cell of interest, and no additional carriers (beads or reporter cells) for capturing analytes are needed, which dramatically improves the availability of microdroplets. This single-cell analytical platform can be applied for determining other secreted cytokines at the single-cell level by changing other immune pairs, which will be an available tool for exploring single-cell metabonomics.

Microfluidic Droplet-SERS Platform for Single-Cell Cytokine Analysis via a Cell Surface Bioconjugation Strategy.

A microfluidic-based surface-enhanced Raman scattering (SERS) platform for analyzing cytokines secreted by single cells is reported based on the elaborate bioconjugation of the immuno-sandwich complex on the probed cell surface. This platform integrates the dual functions of microfluidic droplet separation of single cells and SERS measurement. Two immune nanoprobes (capture probe and SERS probe) are introduced into a microfluidic droplet along with a single cell. They were anchored to the cell membrane protein surface by capturing secreted cytokines to form an immune sandwich structure, realizing the enrichment effect of cytokines above the cell membrane surface and the amplification effect of SERS detection probes. This single-cell analytical platform was applied to track specific cell-secreted vascular endothelial growth factor (VEGF) of different cell lines (MCF-7, SGC, and T24), and highly sensitive detection of VEGF was achieved. Chemometric methods (principal component analysis and t-distributed stochastic neighbor embedding) were adopted for the SERS data analysis, and the support vector machine (SVM) discriminant model was established to test the data. These chemometric methods successfully identify significant differences in the secreting ability of cytokines among three kinds of cancer cell lines, revealing cell heterogeneity. In addition, the behavior of single cells secreting VEGF was monitored time-dependently and was shown to increase with time. This work demonstrates the importance of tracking specific cells secreting cytokines based on the cell surface bioconjugation strategy. Our developed platform provides guidelines for using the single-cell exocytosis factors as biomarkers to assess the early diagnosis of cancer and provide physiological cues for learning single-cell secretions.

Electrostimulus-associated PD-L1 expression on cell membrane revealed by immune SERS nanoprobes.

Programmed cell death ligand 1 (PD-L1) is considered a major immune checkpoint protein that mediates antitumor immune suppression and response. Effectively regulating PD-L1 expression and dynamic monitoring has become a significant challenge in immunotherapy. Herein, we adopted smart surface-enhanced Raman scattering (SERS) nanoprobes to discriminate and monitor the dynamic expression of PD-L1 under external electrostimulation (ES). The PD-L1 expression levels in three cell lines (MCF-7 cells, HeLa cells, and H8 cells) were assessed before and after ES. The results reveal that ES could effectively and rapidly mediate a transformation in the PD-L1 content (or activity) on the cell membrane. Moreover, the molecular profiles of the cell membrane before and after ES were revealed by using the label-free SERS method with the help of immune plasmonic nanoparticles. The cell membrane protein information presented identifiable conformation changes after ES, showing a significant inhibitory effect on the bridge of PD-L1 and its antibody. This study indicates that ES is superior to chemical drugs due to lesser side effects because ES-based regulation does not depend on intracellular signalling pathways. This strategy is versatile and robust for discriminating and monitoring PD-L1 on cell membranes, thus providing potential clinical application value to PD-L1-mediated systems. This study also offers a practical way to assess the molecular profiles of cell membrane proteins in the presence of an external stimulus, which may be applicable to many membrane protein-related studies.

Electrostimulus-triggered reactive oxygen species level in organelles revealed by organelle-targeting SERS nanoprobes.

Electrostimulation (ES) is an important therapeutic method for diseases caused by abnormal intracellular electrical activity. Also, it can induce apoptosis of cells, which is a potential tumor treatment method. At present, there are no relevant studies on changes in intracellular reactive oxygen species (ROS) levels produced in the process of ES, or on the effects of simultaneous implementation of conventional antioxidant inhibitor drugs and ES therapy. To reveal these, two organelle-targeting core-shell plasmonic probes were designed for measuring ROS produced during ES. The probes were delivered into target organelles (nucleus and mitochondrion) before the cells were electrically stimulated for different periods of time. Surface-enhanced Raman scattering (SERS) signals were detected in situ, and the sensing mechanism for the quantitative analysis of ROS is based on the signal reduction of SERS caused by the ROS-etching effect on the silver shell. The detection results revealed that ES could trigger ROS generation in cells, and the ROS levels localized around organelles were assessed by SERS. This study has great potential for exploring abnormal organelle microenvironments via organelle-targeting probes combined with SERS technology.

Tunable multiple light emissions of core-shell structures based on rare earth ions doped on the surfaces of organic cocrystals.

The charge transfer (CT) interactions play a vital role in tuning the luminescence of organic crystals. The enhanced energy transfer (ET) effect in rare earth (RE) ions is a significant method to achieve long-lifetime fluorescence. These studies are of great significance in the fields of photoelectric functional materials. However, the effect of CT interactions on the process of ET from the cocrystal ligand group to RE ions is unknown. In this work, we have doped Eu3+ ions, Tb3+ ions and Eu3+/Tb3+ mixed ions on the surfaces of Phen-TCNB (Phen = 1,10-phenanthroline, and TCNB = 1,2,4,5-tetracyanobenzene) to construct organic cocrystal-type core-shell structures by the epitaxial growth method. The core-shell structures exhibited multiple photoluminescence depending on the types and proportions of RE3+ ions that are doped on the surfaces of the cocrystals. Experimental and theoretical investigations prove that the ET enhancements from ligand groups to Eu3+ ions originate from appropriate energy differences between the lowest triplet states of Phen-TCNB and the lowest excited state of RE3+ ions. In contrast, the reduced Tb3+ 5D4 lifetime is caused by the energy back transfer process since the energy difference becomes small. These results reveal that the multiple luminescences of the cocrystal-type core-shell structures can be adjusted by the CT and ET, and this study provides a new strategy for developing novel optoelectronic materials.

Safety, Tolerability, Pharmacokinetics, and Immunogenicity of a Monoclonal Antibody (SCTA01) Targeting SARS-CoV-2 in Healthy Adults: a Randomized, Double-Blind, Placebo-Controlled, Phase I Study.

SCTA01 is a novel monoclonal antibody with promising prophylactic and therapeutic potential for COVID-19. This study aimed to evaluate the safety, tolerability, pharmacokinetics (PK) and immunogenicity of SCTA01 in healthy adults. This was a randomized, double-blind, placebo-controlled, dose escalation phase I clinical trial. Healthy adults were randomly assigned to cohort 1 (n = 5; 3:2), cohort 2 (n = 8; 6:2), cohort 3, or cohort 4 (both n = 10; 8:2) to receive SCTA01 (5, 15, 30, and 50 mg/kg, respectively) versus placebo. All participants were followed up for clinical, laboratory, PK, and immunogenicity assessments for 84 days. The primary outcomes were the dose-limiting toxicity (DLT) and maximal tolerable dose (MTD), and the secondary outcomes included PK parameters, immunogenicity, and adverse events (AE). Of the 33 participants, 18 experienced treatment-related AEs; the frequency was 52.0% (13/25) in participants receiving SCTA01 and 62.5% (5/8) in those receiving placebo. All AEs were mild. There was no serious AE or death. No DLT was reported, and the MTD of SCTA01 was not reached. SCTA01 with a dose range of 5 to 50 mg/kg had nearly linear dose-proportional increases in Cmax and AUC parameters. An antidrug antibody response was detected in four (16.0%) participants receiving SCTA01, with low titers, between the baseline and day 28, but all became negative later. In conclusion, SCTA01 up to 50 mg/kg was safe and well-tolerated in healthy participants. Its PK parameters were nearly linear dose-proportional. (This study has been registered at ClinicalTrials.gov under identifier NCT04483375.).

Investigating Lysosomal Autophagy via Surface-Enhanced Raman Scattering Spectroscopy.

Autophagy plays a critical role in many vitally important physiological and pathological processes, such as the removal of damaged and aged organelles and redundant proteins. Although autophagy is mainly a protective process for cells, it can also cause cell death. In this study, we employed in situ and ex situ surface-enhanced Raman scattering (SERS) spectroscopies to obtain chemical information of lysosomes of HepG2 cells. Results reveal that the SERS profiles of the isolated lysosomes are different from the in situ spectra, indicating that lysosomes lie in different microenvironments in these two cases. We further investigated the molecular changes of isolated lysosomes according to the autophagy induced by starvation via ex situ SERS. During autophagy, the conformation of proteins and the structures of lipids have been affected, and autophagy-related molecular evidence is given for the first time in the living lysosomes. We expect that this study will provide a reference for understanding the cell autophagy mechanism.

DNA-Mediated Au-Au Dimer-Based Surface Plasmon Coupling Electrochemiluminescence Sensor for BRCA1 Gene Detection.

Herein, we constructed a DNA-mediated Au-Au dimer-based surface plasmon coupling electrochemiluminescence (SPC-ECL) sensor. In the SPC-ECL sensing system, graphite phase carbon nitride quantum dots (GCN QDs) worked as an ECL emitter. A DNA rigid chain structure was employed to connect two Au NPs in an equilateral triangle configuration to form the Au-Au dimers. Due to the hot spot effect, the designed Au-Au dimers had a strong electromagnetic field intensity, which can greatly enhance the ECL signal of GCN QDs than a single Au nanoparticle. The gap distance of dimers can be effectively regulated by the DNA length, which resulted in different electromagnetic field intensities. Therefore, the different SPC-ECL amplification effects on the GCN QD signal by Au-Au dimers have been revealed. The maximum ECL signal of GCN QDs can be enhanced fourfold based on the Au-Au dimers with a gap distance of 2 nm. Furthermore, the biosensor showed good analytical performance for the detection of breast cancer susceptibility gene 1 (BRCA1 genes) (1 fM-1 nM) with a detection limit of 0.83 fM. This work provided an effective and precise SPC-ECL sensing mode for the diagnosis and prognosis of breast cancer.

Polarization-Resolved Electrochemiluminescence Sensor Based on the Surface Plasmon Coupling Effect of a Au Nanotriangle-Patterned Structure.

This work focused on the construction of a nanomaterial-patterned structure for high-resolved ECL signal modulation. Due to the surface coupling effect, the different shapes and distribution states of surface plasmonic nanomaterials not only affect the luminescence intensity enhancement but also decide the electrochemiluminescence (ECL) polarization characteristics. Herein, tin disulfide quantum dots were synthesized via a solvothermal method as ECL emitters. Compared with other nanostructures, Au nanotriangle (Au NT) displayed both the localized surface plasmon resonance electromagnetic enhancement effect and the tip amplification effect, which had significant hot spot regions at three sharp tips. Therefore, self-assembled Au NT-based patterned structures with high density and uniform hot spots were constructed as ideal surface plasmonic materials. More importantly, the distribution states of the hot spots affect the polarization characteristics of ECL, resulting in directional ECL emission at different angles. As a result, a polarization-resolved ECL biosensor was designed to detect miRNA 221. Moreover, this polarization-resolved biosensor achieved good quantitative detection in the linear range of 1 fM to 1 nM and showed satisfactory results in the analysis of the triple-negative breast cancer patients' serum.

Ultrasensitive detection of trypsin in serum via nanochannel device.

A highly sensitive trypsin sensing system in serum was developed by using an anodic alumina oxide (AAO)-based, trypsin substrate-decorated hybrid ion permeation membrane. Owing to the trypsin-triggered peptide hydrolyzation reaction, the surface electrical feature of the peptide-decorated hybrid ion membrane changed. The electric double layer effect reduces the effective ion current diameter in the AAO nano unit, so that the ion current rectification ratio will be enhanced, realizing the quantitative detection of trypsin. The lowest detection concentration can be achieved as low as 0.1 pM. This method is no need for sample pre-preparation, easy to operate, highly sensitive, and also applicable to other enzyme evaluation systems by changing corresponding substrates. This study provides a new idea for selective measurements of proteases in complex biological samples.

Revealing Mitochondrial Microenvironmental Evolution Triggered by Photodynamic Therapy.

Mitochondrion is one of the most important organelles and becomes a target in many cancer therapeutic strategies. Mitochondrial microenvironments in response to therapeutic methods are the key to understand therapeutic mechanisms. However, they are almost rarely studied. Herein, the mitochondrial microenvironments, including mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) after different photodynamic therapy (PDT) dosages, were monitored by fluorescent imaging and compared among three cell lines (HepG2, MCF-7, and LO2). Furthermore, the fluctuations of intramitochondrial pHs were revealed via a plasmonic mitochondrion-targeting surface-enhanced Raman scattering (SERS) pH nanosensor. Results indicate that the MMP decreases gradually with the ROS generation and the cancerous cells exhibit less response to excess ROS relative to normal cells. On the other hand, the pH value in the mitochondria decreases initially and then increases when the amount of ROS increases. The LO2 cell is preliminarily evidenced to have a higher self-adjustment ability due to its better tolerance to differential intra/extracellular pHs. This study may provide a basis for an in-depth understanding of the mechanisms of the mitochondrial targeting-based PDT therapeutic processes. It is also helpful for more accurate and useful diagnosis according to intramitochondrial microenvironments and improvement on therapy efficiency of cancers.