RESUMEN

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Asunto(s)

RESUMEN

Particle plasmon resonance (PPR), or localized surface plasmon resonance (LSPR), utilizes intrinsic resonance in metal nanoparticles for sensor fabrication. While diffraction grating waveguides monitor bioaffinity adsorption with out-of-plane illumination, integrating them with PPR for biomolecular detection schemes remains underexplored. This study introduces a label-free biosensing platform integrating PPR with a diffraction grating waveguide. Gold nanoparticles are immobilized on a glass slide in contact with a sample, while a UV-assisted embossed diffraction grating is positioned opposite. The setup utilizes diffraction in reflection to detect changes in the environment's refractive index, indicating biomolecular binding at the gold nanoparticle surface. The positional shift of the diffracted beam, measured with varying refractive indices of sucrose solutions, shows a sensitivity of 0.97 mm/RIU at 8 cm from a position-sensitive detector, highlighting enhanced sensitivity due to PPR-diffraction coupling near the gold nanoparticle surface. Furthermore, the sensor achieved a resolution of 3.1 × 10-4 refractive index unit and a detection limit of 4.4 pM for detection of anti-DNP. The sensitivity of the diffracted spot was confirmed using finite element method (FEM) simulations in COMSOL Multiphysics. This study presents a significant advancement in biosensing technology, offering practical solutions for sensitive, rapid, and label-free biomolecule detection.

Asunto(s)

RESUMEN

The quality and authenticity of milk are of paramount importance. Cow milk is more allergenic and less nutritious than ewe, goat, or donkey milk, which are often adulterated with cow milk due to their seasonal availability and higher prices. In this work, a silicon photonic dipstick sensor accommodating two U-shaped Mach-Zehnder Interferometers (MZIs) was employed for the label-free detection of the adulteration of ewe, goat, and donkey milk with cow milk. One of the two MZIs of the chip was modified with bovine κ-casein, while the other was modified with bovine serum albumin to serve as a blank. All assay steps were performed by immersion of the chip side where the MZIs are positioned into the reagent solutions, leading to a photonic dipstick immunosensor. Thus, the chip was first immersed in a mixture of milk with anti-bovine κ-casein antibody and then in a secondary antibody solution for signal enhancement. A limit of detection of 0.05% v/v cow milk in ewe, goat, or donkey milk was achieved in 12 min using a 50-times diluted sample. This fast, sensitive, and simple assay, without the need for sample pre-processing, microfluidics, or pumps, makes the developed sensor ideal for the detection of milk adulteration at the point of need.

Asunto(s)

RESUMEN

BACKGROUND: Boar semen quality emphasizes three major criteria: sperm concentration, motility, and morphology. Methods to analyze concentration and motility quickly and objectively readily exist, but few exist for analyzing morphology outside of subjective manual counting. Other vital factors for fertilization, like acrosome health, lack efficient detection methods due to limitations in detection by the human eye and costly biomarker analysis, which is rarely used in semen diagnostics. OBJECTIVE: To overcome these challenges, we propose a novel approach integrating deep-learning technology with high-throughput image-based flow cytometry (IBFC) for objective and accurate analysis of both morphology and label-free acrosome health of thousands of individual spermatozoa at once, as opposed to manually counting on a microscope slide. MATERIALS AND METHODS: Images of 10,000 spermatozoa were captured using an IBFC and manually annotated based on the primary morphological defect or acrosome health status for the training of the convolutional neural network (CNN). The CNN used these images to train and then applied that training to unannotated images to predict the model accuracy. RESULTS: Using the CNNs, high F1 scores of 96.73%, 98.55%, and 99.31% for 20x, 40x, and 60x magnifications, respectively, for morphological classification were attained. Additionally, the model demonstrates an F1 score of 99.8% in detecting subtle acrosome health variations at the 60x magnification. DISCUSSION AND CONCLUSIONS: We have established an integrated approach to rapidly collect and classify morphological defects and acrosome health status, without the use of manual counting or biomarker labeling. Our study underscores the potential of artificial intelligence in semen diagnostics, reducing technician variability, streamlining assays, and facilitating the development of additional label-free detection methods. This innovative approach addresses the barriers hindering biomarker adoption in semen analysis, offering a promising avenue for enhancing reproductive health assessments.

RESUMEN

Field-effect transistor (FET) biosensors based on nanomaterials are promising in the areas of food safety and early disease diagnosis due to their ultrahigh sensitivity and rapid response. However, most academically developed FET biosensors lack real-world reproducibility and comprehensive methodological validation to meet the standards of regulatory bodies. Here, highly uniform and well-packaged semiconducting carbon nanotube (CNT) FET biosensor chips were developed and assessed for the plug-and-play sensing for the rapid and highly sensitive detection of aflatoxin B1 (AFB1) in real food samples to meet international standards. In order to meet the requirements for reproducibility and stability, a scalable residual-free passivation and packaging process was developed for CNT FET biosensors. Portable detection systems were then constructed for on-site detection. The resulting packaged chips were functionalized with nucleic aptamers to enable highly selective detection of AFB1 in food samples with a detection limit (LOD) of 0.55 fg/mL (standard) for AFB1 and cross-reactivity coefficients to interferences as low as 1.8 × 10-7 in simulated solutions. Utilizing the portable detection system, on-site real food detection was achieved with a rapid response time less than 60 s, and LOD of 0.25 pg/kg (standard) in complex corn sample matrices. Single-blind tests demonstrated the ability of the chips to detect AFB1-positive food with 100% accuracy, using a set of 30 peanut samples. Validation experiments confirmed that the detection range, stability, and repeatability met international standards. This study showcased the accuracy, reliability, and potential practical applications of CNT FET biosensor chips in areas such as food safety and rapid biomedical testing.

RESUMEN

Small-molecule biomarkers are ubiquitous in biological fluids with pathological implications, but major challenges persist in their quantitative analysis directly in complex clinical samples. Herein, a molecular-sieving label-free surface-enhanced Raman spectroscopy (SERS) biosensor is reported for selective quantitative analysis of trace small-molecule trimetazidine (TMZ) in clinical samples. Our biosensor is fabricated by decorating a superhydrophobic monolayer of microporous metal-organic frameworks (MOF) shell-coated Au nanostar nanoparticles on a silicon substrate. The design strategy principally combines the hydrophobic surface-enabled physical confinement and preconcentration, MOF-assisted molecular enrichment and sieving of small molecules, and sensitive SERS detection. Our biosensor utilizes such a "molecular confinement-and-sieving" strategy to achieve a five orders-of-magnitude dynamic detection range and a limit of detection of ≈0.5 nM for TMZ detection in either urine or whole blood. We further demonstrate the applicability of our biosensing platform for longitudinal label-free SERS detection of the TMZ level directly in clinical samples in a mouse model.

Asunto(s)

RESUMEN

The sensitive detection of cancer biomarkers is crucial for early accurate diagnostics and therapy of cancer patients. Carcinoembryonic antigen (CEA) is a tumor-associated antigen derived from colon cancer and embryonic tissues. In this study, we have developed a label-free fluorescence biosensing platform for the quantification of CEA with the "turn-on" signal output. This platform employs a label-free strategy that incorporates an aptamer-modified gold nanoparticle (Apt@AuNP) probe for the recognition of CEA, in combination with hybridization chain reaction (HCR) amplification. In the presence of target CEA, Apt@AuNPs selectively capture CEA, resulting in a reduction of subsequent complementary chains (CP) binding on Apt@AuNPs. The remaining CP, acting as the initiator sequence for HCR, triggers the HCR, leading to the formation of abundant G-quadruplex structures. By employing Thioflavin T (ThT) for the formation of G-quadruplex/ThT complexes, the biosensor exhibits a significant enhancement of the fluorescence signal. Under optimized conditions, the biosensor platform demonstrates a limit of detection of 0.03 nM and a linear range from 0.1 to 2.5 nM. Additionally, the specificity investigation reveals the high selectivity of this fluorescent biosensor. Finally, the performance of this method has been validated by successfully detecting CEA in real-life samples, highlighting its potential for clinical applications.

Asunto(s)

RESUMEN

The COVID-19 epidemic and its continuous spread pose a serious threat to public health. Coronavirus strains known as SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) variants have undergone genomic changes. The severity of the symptoms, the efficiency of vaccinations, and the transmission capacity of the virus can be impacted by these alterations. Point-of-care diagnostic assays can identify particular genetic or protein sequences that are exclusive to each variety. Currently, ultrafast, responsive, and accurate antibody detection faces several challenges. Here, we outline the fabrication, implementation, and sensing performance benchmarking of an ultrafast (5 s) and inexpensive (0.15 USD) assay with label-free sensing of SARS-CoV-2 S (Spike)/N (Nucleocapsid) protein and other variants in real patient samples. A label-free DNA aptameric capacitive bio-sensing device was used to detect SARS-CoV-2 variants. Our novel, cutting-edge bio-sensing device contains a Wooden quoits conformation structural aptamer (WQCSA)-based inter-digitated capacitor electronic (WQCSA-IDCE) system. WQCSA-aptamer was used as a switch-turn on response to achieve ultrasensitivity in the variable area of the SARS-CoV-2. The molecular beacon (MB) method was also used to measure the fluorescently colored SARS-CoV-2 S/N protein. These sensors can be used with several types of label-free DNA aptamers to act as rapid, affordable, and label-free biosensors for a variety of critical acute respiratory virus syndrome disorders.

RESUMEN

CRISPR/Cas12a has been widely used in molecular diagnostics due to its excellent trans-cleavage activity. However, conventional reporters, such as F/Q-labeled single-stranded DNA (ssDNA) reporters, enzyme-labeled reporters, and spherical nucleic acid reporters, require complex modification or labeling processes. In this study, we have developed a rapid, universal, and label-free CRISPR/Cas12a-based biomarker detection platform via designing a G-quadruplex (G4) containing a hairpin structure as the reporter. The hairpin loop design of hairpin G4 improves the cleavage efficiency of Cas12a and the signal strength of the G4 binding ligand. Meanwhile, the incorporation of a G4 binding dye (protoporphyrin IX) eliminates the need for complex modifications. The CRISPR-hairpin G4 detection platform is capable of detecting ssDNA, double-stranded DNA, genetic RNAs, and miRNAs. Moreover, this platform achieves label-free detection in clinical samples, demonstrating its practical applicability and efficiency.

RESUMEN

Potency assessment of monoclonal antibodies or corresponding biosimilars in cell-based assays is an essential prerequisite in biopharmaceutical research and development. However, cellular bioassays are still subject to limitations in sample throughput, speed, and often need costly reagents or labels as they are based on an indirect readout by luminescence or fluorescence. In contrast, whole-cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has emerged as a direct, fast and label-free technology for functional drug screening being able to unravel the molecular complexity of cellular response to pharmaceutical reagents. However, this approach has not yet been used for cellular testing of biologicals. In this study, we have conceived, developed and benchmarked a label-free MALDI-MS based cell bioassay workflow for the functional assessment of complement-dependent cytotoxicity (CDC) of Rituximab antibody. By computational evaluation of response profiles followed by subsequent m/z feature annotation via fragmentation analysis and trapped ion mobility MS, we identified adenosine triphosphate and glutathione as readily MS-assessable metabolite markers for CDC and demonstrate that robust concentration-response characteristics can be obtained by MALDI-TOF MS. Statistical assay performance indicators suggest that whole-cell MALDI-TOF MS could complement the toolbox for functional cellular testing of biopharmaceuticals.

Asunto(s)

RESUMEN

BACKGROUND: Mushroom poisoning poses a significant global health concern, with high morbidity and mortality rates. The primary lethal toxins responsible for this condition are alpha-amanitin (ɑ-AMA) and beta-amanitin (ß-AMA). As a promising bio-recognition molecules in biosensors, aptamers, have been broadly used in the field of food detection. However, the current SELEX-based methods for screening aptamers for structurally similar small molecules were limited by the labelling or salt ion induction. In this study, we aimed to develop a novel label-free SELEX strategy for the screening of aptamers with high affinity and constructed new aptasensors for the detection of ɑ-AMA and ß-AMA. RESULTS: A novel label-free SELEX strategy based on the positively charged gold nanoparticles (AuNPs) was proposed to simultaneous screening of aptamers for ɑ-AMA and ß-AMA. Only 18 rounds of SELEX were required to obtain new aptamers. The candidate aptamers were analyzed by colloidal gold assay, and the sequences of ɑ-30 and ß-37 displayed great affinity with Kd values of 22.26 nM and 23.32 nM, respectively, without interference from botanical toxins. Notably, the truncated aptamers ɑ-30-2 (50 bp) and ß-37-2 (57 bp) exhibited higher affinity than their original counterpart (79 bp). Subsequently, the selected aptamers were utilized to construct recognition probes for electrochemical aptasensors based on hairpin cyclic cleavage of substrates by Cu2+ dependent DNAzyme and Exo I-triggered recycling cascades. The detection platform showed excellent analytical performance with limits of detection as low as 4.57 pg/mL (ɑ-AMA) and 8.49 pg/mL (ß-AMA). Moreover, the aptasensors exhibited superior performance in mushroom and urine samples. SIGNIFICANCE: This work developed a simple and efficient label-free SELEX method for screening new aptamers for ɑ-AMA and ß-AMA, which employed the positively charged AuNPs as the screening medium, without the need for chemical labelling of libraries or induction of salt ions. Furthermore, two novel electrochemical aptasensors were developed based on our newly obtained aptamers, which offer the new biosensing tool for ultrasensitive detection of the AMA poisoning, showing great potential in practical applications.

Asunto(s)

RESUMEN

In this work, a boric-acid-modified Fe3O4@Au@BA-MOF composite material as a multifunctional SERS substrate was ingeniously constructed for detecting both pathogens and antibiotics as well as photothermally inactivating the pathogens. Through improving the dispersity and stability of gold nanoparticles (Au NPs), leveraging the specificity of boric acid (BA) groups in recognizing cis-diol structures, and the ability of SERS technology to provide unique fingerprint spectra of targets, the sensitive and stable detection of pathogens and antibiotics was achieved. Compared with Au NPs and Fe3O4@Au, the SERS enhancement factor of Fe3O4@Au@BA-MOF was 4.31 × 106, which was about 400 times and 16 times higher than the former two, respectively. Among the existing work, the limit of detection for pathogens was lower or comparable, and it exhibited good stability, maintaining consistent performance for 23 days. Additionally, this substrate achieved efficient photothermal inactivation of pathogens under both near-infrared light and natural light excitation. Within 8 min of near-infrared light irradiation, the bactericidal rates for Staphylococcus aureus and Escherichia coli reach 100% and 99.3%, respectively.

RESUMEN

Live-cell label-free imaging of a microscopic biological barrier, generally referred to as 'tight junction', was realized by a recently developed electric-double-layer modulation imaging (EDLMI). The method allowed quantitative imaging of barrier integrity in real time, thus being an upper compatible of transepithelial electrical resistance (TEER) which is a conventional standard technique to evaluate spatially averaged barrier integrity. We demonstrate that the quantitative and real-time imaging capability of EDLMI unveils fundamental dynamics of biological barrier, some of which are totally different from conventional understandings.

Asunto(s)

RESUMEN

Scalable electronic devices that can detect target biomarkers from clinical samples hold great promise for point-of-care nucleic acid testing, but still cannot achieve the detection of target molecules at an attomolar range within a short timeframe (<1 h). To tackle this daunting challenge, we integrate graphene field-effect transistors (GFETs) with exponential target recycling and hybridization chain reaction (TRHCR) to detect oligonucleotides (using miRNA as a model disease biomarker), achieving a detection limit of 100 aM and reducing the sensing time by 30-fold, from 15 h to 30 min. In contrast to traditional linear TRHCR, our exponential TRHCR enables the target miRNA to initiate an autocatalytic system with exponential kinetics, significantly accelerating the reaction speed. The resulting reaction products, long-necked double-stranded polymers with a negative charge, are effectively detected by the GFET through chemical gating, leading to a shift in the Dirac voltage. Therefore, by monitoring the magnitude of this voltage shift, the target miRNA is quantified with high sensitivity. Consequently, our approach successfully detects 22-mer miRNA at concentrations as low as 100 aM in human serum samples, achieving the desired short timeframe of 30 min, which is congruent with point-of-care testing, and demonstrates superior specificity against single-base mismatched interfering oligonucleotides.

Asunto(s)

RESUMEN

Heme released from damaged and senescent red blood cells (RBCs) may contribute to oxidant-mediated cell injury. One of the recently investigated physiological processes, essential in preventing the inflammatory impact of labile heme, is its uptake from the bloodstream by endothelial cells (ECs). In this study, we investigated heme uptake by ECs starting from the model studies on the in vitro cellular level, through the endothelium layer on the ex vivo murine aortic tissues. As the cellular model, Human Aortic Endothelial Cells (HAECs) were chosen, and the concentration of labile heme was adjusted so to avoid the excessive toxic effect of the labile heme. We utilized label-free Raman imaging with two different excitation wavelengths to capture the uptake process in situ and characterize the oxidation state of the iron ion in the intercalated heme. The phenomenon of heme uptake was demonstrated in both, the healthy control C57Bl/6J and FVB animals, as well as in mice with developed atherosclerosis (ApoE/LDLR-/- mice). In the presented work, we presented for the first time Raman-based evidence on the heme uptake process by endothelial cells in both, in vitro and ex vivo systems.

Asunto(s)

RESUMEN

Workplace exposure to diisocyanates like 4,4'-methylene diphenyl diisocyanate can cause occupational asthma (MDI-OA), and the underlying biological pathways are still being researched.Although uncertainty remains, evidence supports the hypothesis that dermal exposure to MDI plays an important role in the development of MDI-OA.Gene expression, proteomics, and informatics tools were utilized to characterize changes in expression of RNA and protein in cultured human HEKa keratinocyte cells following exposure to conjugates of MDI with glutathione (MDI-GSH).RT-qPCR analysis using a panel of 39 candidate primers demonstrated 9 candidate genes upregulated and 30 unchanged.HPLC-MS/MS analysis of HEKa cell lysate identified 18,540 proteins across all samples Sixty proteins demonstrate statistically significant differential expression in exposed cells, some of which suggest activation of immune and inflammatory pathways.The results support the hypothesis that dermal exposures have the potential to play an important role in the development of MDI-OA. Furthermore, proteomic and gene expression data suggest multiple immune (adaptive and innate) and inflammatory pathways may be involved in the development of MDI-OA.

RESUMEN

Integrating plant proteins into meat products offers a sustainable way to reduce the environmental impact of meat consumption while satisfying the growing flexitarian population. This study explored the effects of textured vegetable proteins (TVPs) on the physico-chemical attributes and flavour profile of hybrid salamis using 4D label-free proteomics. Results showed that hybrid salamis had lower pH, reduced water activity and increased weight loss compared with traditional salamis, along with greater hardness and a slightly rough, porous texture with a filamentous structure. TVPs substantially modified crucial meaty flavour compounds (nitrogen oxides, sulfides and pyrazine), increasing heightening sourness and bitterness while diminishing umami. Proteomic analysis revealed significant upregulation of myosin and actin in hybrid salamis; notably, these proteins were involved in glycerol-3-phosphate dehydrogenase activity and calcineurin-mediated signalling, underscoring their role in flavour enhancement. Therefore, hybrid salamis offer an attractive alternative to traditional salamis by merging meat-like taste and texture with plant protein.

RESUMEN

Large yellow croaker (Larimichthys crocea) is susceptible to oxidative denaturation during storage. This work is to investigate the quality alterations by analyzing its physicochemical changes and proteomics throughout preservation under refrigeration, frozen, and slurry ice (SI) conditions. Results revealed that the freshness of large yellow croaker, as evaluated by indicators such as total volatile basic nitrogen, total viable count, and thiobarbituric acid reactive substances, was well maintained while stored in the SI group. Meanwhile, the water distribution in the muscle tissue of group SI exhibited slower fluctuations, thereby preserving the integrity of fish muscle cells. Based on label-free proteomic analysis, a considerable downregulation was observed in the mitogen-activated protein kinase (MAPK) signaling pathway, indicating that SI decelerated this metabolic pathway and effectively delayed the deterioration of muscle. Therefore, the application of SI provides potential for maintaining the quality stability of large yellow croaker.

RESUMEN

In this study, we adapted an HP D100 Single Cell Dispenser - a novel low-cost thermal inkjet (TIJ) platform with impedance-based single cell detection - for dispensing of individual cells and one-pot sample preparation. We repeatedly achieved label-free identification of up to 1,300 proteins from a single cell in a single run using an Orbitrap Fusion Lumos Mass Spectrometer coupled to either an Acquity UPLC M-class system or a Vanquish Neo UHPLC system. The developed sample processing workflow is highly reproducible, robust, and applicable to standardized 384- and 1536-well microplates, as well as glass LC vials. We demonstrate the applicability of the method for proteomics of single cells from multiple cell lines, mixed cell suspensions, and glioblastoma tumor spheroids. As additional proof of robustness, we monitored the results of genetic manipulations and the expression of engineered proteins in individual cells. Our cost-effective and robust single-cell proteomics workflow can be transferred to other labs interested in studying cells at the individual cell level.

RESUMEN

Rapid virus identification is crucial for preventing outbreaks. The COVID-19 pandemic has highlighted the critical nature of rapid virus detection. Here, we designed a label-free electrochemical biosensor modified with gold nanoparticles (AuNPs) to detect IgG antibodies from human serum, enabling rapid point-of-care diagnostics. AuNPs were synthesized and characterized. A multivariate optimization was carried out to determine the optimal condition for functionalizing AuNPs with anti-IgG. Subsequently, using a glassy carbon electrode (GCE), a modified AuNPs/GCE electrochemical biosensor was developed for IgG detection. The results indicated that AuNPs displayed a spherical morphology with a size distribution of 19.54 nm. Additionally, the zeta potential was recorded at -7.84 mV. Central composite design (CCD) analysis determined the optimal conditions for functionalizing AuNPs to be an anti-IgG concentration of 320 µg mL-1, a temperature of 25 °C, and pH of 7.4. The characterization study confirmed the successful synthesis and functionalization of AuNPs. Through electrochemical impedance spectroscopy measurement, the biosensor demonstrated a limit of detection (LOD) of 0.2 ng mL-1 and limit of quantification (LOQ) of 0.8 ng mL-1. Furthermore, tests in real samples showed the interaction between IgG antibodies in serum samples and AuNPs/GCE, confirming the biosensor's ability to detect and quantify IgG in clinical samples.