Search Results (17)

Background: Staphylococci, recognized for their virulence and antibiotic resistance, are important in both human and veterinary medicine. Loss of sensitivity to beta-lactam antibiotics, such as ampicillin, complicates therapy, prompting the search for alternative antibacterials or ways to increase drug efficacy. Silver and gold nanoparticles (AgNPs, AuNPs) are promising on their own or in combination with antibiotics. Methods: The aim of this study is to compare the biological activity of pure, washed AgNPs and AuNPs with biosynthesized nanoparticles from Alchemilla vulgaris (AgNPs-Av and AuNPs-Av). Their antibacterial, antibiofilm, and biofilm-eradication effects on the tested antibiotic-resistant, biofilm-forming staphylococci (Methicillin-resistant Staphylococcus aureus (MRSA) and multiresistant Non-aureus staphylococci and mammaliicocci (NASM)) were evaluated using in vitro microdilution methods. Results: AgNPs-Av and AuNPs-Av inhibited bacterial growth at 50 μg/mL, while a significant suppression of biofilm formation was observed at just 25 μg/mL. Our research showed that neither AuNPs-Av nor AuNPs disrupts bacterial biofilm. AgNPs-Av effectively eradicated the biofilm at 50 μg/mL. NPs and ampicillin at subinhibitory antibiotic concentrations against the tested staphylococci. The results showed significant antibacterial and antibiofilm effects (p = 0.001). Partially, biofilm-eradication activity and strong antibiotic potentiation were also detected. Conclusions: These findings highlight the importance of rational combination therapy to improve antibiotic effectiveness and reduce bacterial resistance. Full article

In this study, three point mutations of EGFR relevant to lung cancer therapy are detected. Mutated EGFR is the target of a therapy for non-small cell lung cancer (NSCLC) using tyrosine kinase inhibitors (TKIs) as treatment drugs. Background/Objectives: Point mutations in exon 21 (L858R and L861Q) of the EGFR gene are TKI-sensitive; however, mutations in exon 20 (T790M) are TKI-resistant. Therefore, a fast detection method that classifies an NSCLC patient to be drug sensitive or drug resistant is highly clinically relevant. Methods: Probes were designed to detect three point mutations in genomic samples based on DNA hybridization on a solid surface. A method has been developed to detect single nucleotide polymorphism (SNP) for these mutation detections in the 16-channel nanobioarray chip. The wash by gold-nanoparticles (AuNP) was used to assist the differentiation detection. Results: The gold nanoparticle-assisted wash method has enhanced differentiation between WT and mutated sequences relevant to the EGFR sensitivity to tyrosine kinase inhibitors. Conclusions: The WT and mutated sequences (T790M, L858R and L861Q) in genomic samples were successfully differentiated from each other. Full article

This study presents a comparative investigation of plasmonic sensing platforms based on colloidal gold nanoparticle (AuNP) suspensions and gold film over nanosphere (AuFoN) solid substrates for the detection of epidermal growth factor receptor (EGFR), an essential biomarker and therapeutic target in oncology. The strategy relies on fluorescence emission modulation of an Atto647N-labeled DNA oligomer competitively bound to an EGFR-specific aptamer. Our results demonstrate that the colloidal AuNPs can function as competitive binding sensors, leading to fluorescence quenching upon fluorophore attachment to the surface of the NPs and partial fluorescence recovery due to EGFR-induced displacement of the fluorophore–aptamer complex. This specificity was confirmed by reversed binding experiments. However, the system proved highly sensitive to the experimental design: excessive washing (centrifugation) led to unspecific aggregation and signal loss, while reduced washing steps improved signal retention and revealed EGFR-induced fluorophore displacement into the supernatant. On the contrary, film-based substrates exhibited strong initial fluorescence, but failed to retain the fluorophore–aptamer complex after washing, resulting in fluorescence decay independent of EGFR incubation. This indicates that AuFoN lacked the binding stability necessary for specific displacement-based sensing. These findings highlight that while colloidal AuNPs can support competitive binding detection, their reproducibility is limited by colloidal stability and protocol sensitivity, whereas AuFoN substrates require improved surface functionalization strategies. The study emphasizes the critical role of surface chemistry, aptamer–fluorophore affinity, and washing protocols in determining the success or failure of plasmon-enhanced aptamer-based biosensing systems and suggests opportunities for improving specificity and robustness in future designs. Full article

We present a lateral flow aptasensor for the visual detection of alpha-fetoprotein (AFP) in human serum. Leveraging the precise molecular recognition capabilities of aptamers and the distinct optical features of gold nanoparticles, a model system utilizing AFP as the target analyte, along with a pair of aptamer probes, is implemented to establish proof-of-concept on standard lateral flow test strips. It is the first report of an antibody-free lateral flow assay using aptamers as recognition probes for the detection of AFP. The analysis circumvents the numerous incubation and washing steps that are typically involved in most current aptamer-based protein assays. Qualitative analysis involves observing color changes in the test area, while quantitative data are obtained by measuring the optical response in the test zone using a portable strip reader. The biosensor exhibits a linear detection range for AFP concentrations between 10 and 100 ng/mL, with a minimum detection limit of 10 ng/mL. Additionally, it has been successfully applied to detect AFP in human serum samples. The use of aptamer-functionalized gold nanoparticle probes in a lateral flow assay offers great promise for point-of-care applications and fast, on-site detection. Full article

Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including in COVID-19. Currently, lateral flow tests dominate in this area and show good analytical performance. However, studies to improve the effectiveness of such tests remain important. In comparison with lateral flow tests, vertical flow immunoassays allow for a reduction in assay duration and the influence of the hook effect. Additionally, the use of carbon black nanoparticles (CNPs) as a color label can provide a lower detection limit (LOD) compared to conventional colloidal gold. Therefore, we have developed a vertical flow immunoassay for the detection of IgG against SARS-CoV-2 spike protein in human serum samples by applying a conjugate of CNPs with anti-human IgG mouse monoclonal antibodies (CNP@MAb). The vertical flow assay device consists of a plastic cassette with a hole on its top containing a nitrocellulose membrane coated with spike protein and an absorbent pad. The serum sample, washing buffer, and CNP@MAb flow vertically through the nitrocellulose membrane and absorbent pads, reducing assay time and simplifying the procedure. In positive samples, the interaction of CNP@MAb with anti-spike antibodies leads to the appearance of black spots, which can be visually detected. The developed method allows for rapid visual detection (5–7 min) of IgG vs. spike protein, with a LOD of 7.81 BAU/mL. It has been shown that an untrained operator can perform the assay and visually evaluate its results. Thus, the presented assay can be used in the further development of test systems for the serological diagnostics of COVID-19 or post-vaccination immunity monitoring. Full article

Raman spectroscopy is a powerful analytical technique widely used for quantitative and qualitative analysis. However, the development of inexpensive, reproducible, and reusable enhancing substrates remains a challenge for material scientists and analytical chemists. In this study, we address this challenge by demonstrating the deposition of core–shell nanoparticles consisting of a gold core and a thin inert SiO₂ shell within a confined space, resulting in the formation of a highly efficient Raman-enhancing structure. Nanoparticles were characterized by UV–vis spectroscopy, dynamic light scattering, and total reflectance X-ray fluorescence spectroscopy, whereas the prepared substrates were characterized by scanning electron microscopy and Raman spectroscopy with a model molecule, malachite green. The relationship between Raman intensity and the loading of malachite green dye exhibited linearity, indicating the uniform spatial distribution of hotspots across the substrate. The limit of detection was determined as 2.9 μM of malachite green when 10 uL was distributed over a ca. 25 mm² surface area. Moreover, the same substrate, after thorough washing in ethanol, was successfully employed for the detection of bovine serum albumin at a concentration level of 55 μg mL⁻¹, demonstrating its reusability and versatility. Our findings highlight the potential of these substrates for various applications in biomedical research, clinical diagnosis, and beyond. Full article

Hospitals and nursing home wards are areas prone to the propagation of infections and are of particular concern regarding the spreading of dangerous viruses and multidrug-resistant bacteria (MDRB). MDRB infections comprise approximately 20% of cases in hospitals and nursing homes. Healthcare textiles, such as blankets, are ubiquitous in hospitals and nursing home wards and may be easily shared between patients/users without an adequate pre-cleaning process. Therefore, functionalizing these textiles with antimicrobial properties may considerably reduce the microbial load and prevent the propagation of infections, including MDRB. Blankets are mainly comprised of knitted cotton (CO), polyester (PES), and cotton-polyester (CO–PES). These fabrics were functionalized with novel gold-hydroxyapatite nanoparticles (AuNPs-HAp) that possess antimicrobial properties, due to the presence of the AuNPs’ amine and carboxyl groups, and low propensity to display toxicity. For optimal functionalization of the knitted fabrics, two pre-treatments, four different surfactants, and two incorporation processes were evaluated. Furthermore, exhaustion parameters (time and temperature) were subjected to a design of experiments (DoE) optimization. The concentration of AuNPs-HAp in the fabrics and their washing fastness were critical factors assessed through color difference (ΔE). The best performing knitted fabric was half bleached CO, functionalized using a surfactant combination of Imerol^® Jet-B (surfactant A) and Luprintol^® Emulsifier PE New (surfactant D) through exhaustion at 70 °C for 10 min. This knitted CO displayed antibacterial properties even after 20 washing cycles, showing its potential to be used in comfort textiles within healthcare environments. Full article

Since the outbreak of the pandemic respiratory virus SARS-CoV-2 (COVID-19), academic communities and governments/private companies have used several detection techniques based on gold nanoparticles (AuNPs). In this emergency context, colloidal AuNPs are highly valuable easy-to-synthesize biocompatible materials that can be used for different functionalization strategies and rapid viral immunodiagnosis. In this review, the latest multidisciplinary developments in the bioconjugation of AuNPs for the detection of SARS-CoV-2 virus and its proteins in (spiked) real samples are discussed for the first time, with reference to the optimal parameters provided by three approaches: one theoretical, via computational prediction, and two experimental, using dry and wet chemistry based on single/multistep protocols. Overall, to achieve high specificity and low detection limits for the target viral biomolecules, optimal running buffers for bioreagent dilutions and nanostructure washes should be validated before conducting optical, electrochemical, and acoustic biosensing investigations. Indeed, there is plenty of room for improvement in using gold nanomaterials as stable platforms for ultrasensitive and simultaneous “in vitro” detection by the untrained public of the whole SARS-CoV-2 virus, its proteins, and specific developed IgA/IgM/IgG antibodies (Ab) in bodily fluids. Hence, the lateral flow assay (LFA) approach is a quick and judicious solution to combating the pandemic. In this context, the author classifies LFAs according to four generations to guide readers in the future development of multifunctional biosensing platforms. Undoubtedly, the LFA kit market will continue to improve, adapting researchers’ multidetection platforms for smartphones with easy-to-analyze results, and establishing user-friendly tools for more effective preventive and medical treatments. Full article

Extracellular vesicle (EV) research is a rapidly developing field, mainly due to the key role of EVs in intercellular communication and pathophysiological processes. However, the heterogeneity of EVs challenges their exploration and the establishment of gold-standard methods. Here, we aimed to reveal the influence of technical changes on EV biology and the reliability of experimental data. We used B16F1 melanoma cells as a model and applied nanoparticle tracking analysis, mass spectrometry (LC-MS/MS) and pathway enrichment analysis to analyze the quantity, size distribution, proteome and function of their small EVs (sEVs) produced in sEV-depleted fetal bovine serum (FBS)-containing medium or serum-free medium. Additionally, we investigated the effects of minor technical variances on the quality of sEV preparations. We found that storage of the isolates at −80 °C has no adverse effect on LC-MS/MS analysis, and an additional washing step after differential ultracentrifugation has a minor influence on the sEV proteome. In contrast, FBS starvation affects the production and proteome of sEVs; moreover, these vesicles may have a greater impact on protein metabolism, but a smaller impact on cell adhesion and membrane raft assembly, than the control sEVs. As we demonstrated that FBS starvation has a strong influence on sEV biology, applying serum-free conditions might be considered in in vitro sEV studies. Full article

This work compared the effectiveness of several Methylene Blue (MB)-based protocols for photo-eradication of biofilms formed on the surface of the glass and stainless steel discs by S. aureus MRSA isolates using a diode laser (λ = 665 nm; output power 40 mW; energy fluence was 189 J cm⁻²). The results obtained showed that MB alone, up to a concentration of 62.5 mgL⁻¹, had limited photo-bactericidal activity. It was possible to enhance the activity of MB using two types of spherical gold nanoparticles of similar sizes, 15 ± 3 nm/20 ± 3 nm, but differing in the method of their synthesis and stabilization. The enhancement of the photodestruction effect was related to the increased production of hydroxyl radicals by the MB+gold nanoparticles mixture, and this mixture showed dark cytotoxicity against the cocci studied. Effective destruction (mortality above 99.9%) of the biofilms formed by MRSA isolates was also possible without the use of gold nanoparticles, but the concentration of MB had to be at least 125 mgL⁻¹. A highly efficient protocol of photodestruction of biofilms, consisting of triple exposure of biofilms to laser light in the presence of MB alone, combined with the removal of dead bacteria protecting deep layers of pathogens against photosensitization, was also described. Full article

The fabrication of a coffee ring was studied in this work to improve its sensitivity in detecting trace analytes based on surface-enhanced Raman scattering (SERS). Gold nanoparticles were synthesized with diameters of ~40 nm through the sodium citrate reduction method, and rhodamine 6G (R6G) was employed as a probe to evaluate the performance of the fabricated coffee rings. The results showed that the coffee ring formed from the water-washed gold nanoparticles presented more orderly and regular morphology as well as better SERS properties than the unwashed ones. Furthermore, both the concentration and the amount of gold nanoparticles were found to affect its SERS performance. Using the optimized coffee ring as a SERS substrate, trace R6G with a concentration of 5 × 10⁻⁸ M was detected. This sensing platform could realize aflatoxin B1 (AFB1) detection down to 5 × 10⁻⁷ M and was demonstrated to function well in real-sample testing. Full article

A fluorescence off-on method for the detection of Sorghum mosaic virus (SrMV) based on fluorescence resonance energy transfer (FRET) was developed in this study. The biosensor was realized by the adsorption of SrMV coat protein (CP) on gold nanoparticle (AuNP) and conjugation of anti-SrMV antibody (Ab) on cadmium telluride quantum dot (CdTe QD). The optimum quenching efficiency was about 50% obtained at a CdTe QD-Ab/AuNP-CP ratio of 1:7. Moreover, the feasibility of the developed biosensor was verified by the detection of purified CP and the limit of detection was estimated as 0.02 μg/mL. This strategy was also successfully applied to monitor SrMV CP in plant sap with a recovery rate between 97.7 and 107.4%. The developed biosensor is a simple, rapid, and efficient technique which does not need excessive washing and separation steps. Full article

The increasing popularity of nanoparticles in many applications has led to the fact that these persistent materials pollute our environment and threaten our health. An online sensor system for monitoring the presence of nanoparticles in fresh water would be highly desired. We propose a label-free sensor based on SPR imaging. The sensitivity was enhanced by a factor of about 100 by improving the detector by using a high-resolution camera. This revealed that the light source also needed to be improved by using LED excitation instead of a laser light source. As a receptor, different self-assembled monolayers have been screened. It can be seen that the nanoparticle receptor interaction is of a complex nature. The best system when taking sensitivity as well as reversibility into account is given by a dodecanethiol monolayer on the gold sensor surface. Lanthanide-doped nanoparticles, 29 nm in diameter and with a similar refractive index to the most common silica nanoparticles were detected in water down to 1.5 µg mL⁻¹. The sensor can be fully regenerated within one hour without the need for any washing buffer. This sensing concept is expected to be easily adapted for the detection of nanoparticles of different size, shape, and composition, and upon miniaturization, suitable for long-term applications to monitor the quality of water. Full article

Food manufacturers are aiming to manage the levels of cross-contamination of allergens within food processing plants and ultimately move away from precautionary labelling. Hence, the need for rapid methods to detect allergens cross-contamination. A sensitive and selective label-free nanoMIPs based sensor was developed and tested for the detection of β-lactoglobulin (BLG). NanoMIPs were synthesized using solid-phase synthesis and appeared as spherical nanoparticles with sizes ranging from 264–294 nm, using dynamic light scattering (DLS). The nanoMIPs were functionalized with amine groups and attached to the surface of the SPR gold chip via amine-coupling protocol. The SPR nanoMIPs-based sensor demonstrated a detection limit of 3 ng mL⁻¹ (211 pM) over a linear range of 1–5000 ng mL⁻¹, with binding affinity of 7.0 × 10⁻⁸ M and specificity towards BLG. With further testing and final optimization, the developed nanosensor can be integrated on-line or at-line cleaning-in-place (CIP) wash systems, allowing to effectively monitor milk protein allergens as a rapid, point-of-source methodology. Full article

Although immunomagnetic separation is a useful sample pretreatment method that can be used to separate target pathogens from a raw sample, it is challenging to remove unbound free magnetic nanoparticles (MNPs) for colorimetric detection of target pathogens. Here, size-based filtration was exploited for the rapid on-site detection of pathogens separated by immunomagnetic separation in order to remove unbound free MNPs using a finger-powered microfluidic device. A membrane filter and an absorbent pad were integrated into the device and a mixture of unbound free MNPs and MNP-bound Escherichia coli (E. coli) O157:H7 was dispensed over the membrane filter by pressing and releasing the pressure chamber. A colorimetric signal was generated by MNP-bound E. coli O157:H7 while unbound free MNPs were washed out by the absorbent. Furthermore, the colorimetric signals can be amplified using a gold enhancer solution when gold-coated MNPs were used instead of MNPs. As a result, 10² CFU/mL E. coli O157:H7 could be detected by the enhanced colorimetric signal on a proposed device. Full article