Rationally designed protein A surface molecularly imprinted magnetic nanoparticles for the capture and detection of Staphylococcus aureus.

Staphylococcus aureus (S. aureus), a commensal organism found on the human skin, is commonly associated with nosocomial infections and exhibits virulence mediated by toxins and resistance to antibiotics. The global threat of antibiotic resistance has necessitated antimicrobial stewardship to improve the safe and appropriate use of antimicrobials; hence, there is an urgent demand for the advanced, cost-effective, and rapid detection of specific bacteria. In this regard, we aimed to selectively detect S. aureus using surface molecularly imprinted magnetic nanoparticles templated with a well-known biomarker protein A, specific to S. aureus. Herein, a highly selective surface molecularly imprinted polymeric thin layer was created on ∼250 nm magnetic nanoparticles (MNPs) through the immobilization of protein A to aldehyde functionalized MNPs, followed by monomer polymerization and template washing. This study employs the rational selection of monomers based on their computationally predicted binding affinity to protein A at multiple surface residues. The resulting MIPs from rationally selected monomer combinations demonstrated an imprinting factor as high as ∼5. Selectivity studies revealed MIPs with four-fold higher binding capacity (BC) to protein A than other non-target proteins, such as lysozyme and serum albumin. In addition, it showed significant binding to S. aureus, whereas negligible binding to other non-specific Gram-negative, i.e. Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Gram-positive, i.e. Bacillus subtilis (B. subtilis), bacteria. This MIP was employed for the capture and specific detection of fluorescently labeled S. aureus. Quantitative detection was performed using a conventional plate counting technique in a linear detection range of 101-107 bacterial cells. Remarkably, the MIPs also exhibited approximately 100% cell recovery from milk samples spiked with S. aureus (106 CFU mL-1), underscoring its potential as a robust tool for sensitive and accurate bacterial detection in dairy products. The developed MIP exhibiting high affinity and selective binding to protein A finds its potential applications in the magnetic capture and selective detection of protein A as well as S. aureus infections and contaminations.

Greening the pathways: a comprehensive review of sustainable synthesis strategies for silica nanoparticles and their diverse applications.

Silica nanoparticles (SiNPs) have emerged as a multipurpose solution with wide-ranging applications in various industries such as medicine, agriculture, construction, cosmetics, and food production. In 1961, Stöber introduced a ground-breaking sol-gel method for synthesizing SiNPs, which carried a new era of exploration both in academia and industry, uncovering numerous possibilities for these simple yet multifaceted particles. Inspite of numerous reported literature with wide applicability, the synthesis of these nanoparticles with the desired size and functionalities poses considerable challenges. Over time, researchers have strived to optimize the synthetic route, particularly by developing greener approaches that minimize environmental impact. By reducing hazardous chemicals, energy consumption, and waste generation, these greener synthesis methods have become an important focus in the field. This review aims to provide a comprehensive analysis of the various synthetic approaches available for different types of SiNPs. Starting from the Stöber' method, we analyze other methods as well to synthesis different types of SiNPs including mesoporous, core-shell and functionalized nanoparticles. With increasing concerns with the chemical methods associated for environmental issues, we aim to assist readers in identifying suitable greener synthesis methods tailored to their specific requirements. By highlighting the advancements in reaction time optimization, waste reduction, and environmentally friendly precursors, we offer insights into the latest techniques that contribute to greener and more sustainable SiNPs synthesis. Additionally, we briefly discuss the diverse applications of SiNPs, demonstrating their relevance and potential impact in fields such as medicine, agriculture, and cosmetics. By emphasizing the greener synthesis methods and economical aspects, this review aims to inspire researchers and industry professionals to adopt environmentally conscious practices while harnessing the immense capabilities of SiNPs.

A facile cost-effective electrolyte-assisted approach and comparative study towards the Greener synthesis of silica nanoparticles.

Nowadays, silica nanoparticles are gaining tremendous importance because of their wide applications across different domains such as drug delivery, chromatography, biosensors, and chemosensors. The synthesis of silica nanoparticles generally requires a high percentage composition of organic solvent in an alkali medium. The eco-friendly synthesis of silica nanoparticles in bulk amounts can help save the environment and is cost-effective. Herein, efforts have been made to minimize the concentration of organic solvents used during synthesis via the addition of a low concentration of electrolytes, e.g., NaCl. The effects of electrolytes and solvent concentrations on nucleation kinetics, particle growth, and particle size were investigated. Ethanol was used as a solvent in various concentrations, ranging from 60% to 30%, and to optimize and validate the reaction conditions, isopropanol and methanol were also utilized as solvents. The concentration of aqua-soluble silica was determined using the molybdate assay to establish reaction kinetics, and this approach was also utilized to quantify the relative concentration changes in particles throughout the synthesis. The prime feature of the synthesis is the reduction in organic solvent usage by up to 50% using 68 mM NaCl. The surface zeta potential was reduced after the addition of an electrolyte, which made the condensation process faster and helped reaching the critical aggregation concentration in a shorter time. The effect of temperature was also monitored, and we obtained homogeneous and uniform nanoparticles by increasing the temperature. We found that it is possible to tune the size of the nanoparticles by changing the concentration of electrolytes and the temperature of the reaction using an eco-friendly approach. The overall cost of the synthesis can also be reduced by ∼35% by adding electrolytes.

COVID-19 Detection Using a 3D-Printed Micropipette Tip and a Smartphone.

The COVID-19 pandemic has caused over 7 million deaths worldwide and over 1 million deaths in the US as of October 15, 2022. Virus testing lags behind the level or availability necessary for pandemic events like COVID-19, especially in resource-limited settings. Here, we report a low cost, mix-and-read COVID-19 assay using a synthetic SARS-CoV-2 sensor, imaged and processed using a smartphone. The assay was optimized for saliva and employs 3D-printed micropipette tips with a layer of monoclonal anti-SARS-CoV-2 inside the tip. A polymeric sensor for SARS-CoV-2 spike (S) protein (COVRs) synthesized as a thin film on silica nanoparticles provides 3,3',5-5'-tetramethylbenzidine responsive color detection using streptavidin-poly-horseradish peroxidase (ST-poly-HRP) with 400 HRP labels per molecule. COVRs were engineered with an NHS-PEG4-biotin coating to reduce nonspecific binding and provide affinity for ST-poly-HRP labels. COVRs binds to S-proteins with binding strengths and capacities much larger than salivary proteins in 10% artificial saliva-0.01%-Triton X-100 (as virus deactivator). A limit of detection (LOD) of 200 TCID50/mL (TCID50 = tissue culture infectious dose 50%) in artificial saliva was obtained using the Color Grab smartphone app and verified using ImageJ. Viral load values obtained in 10% pooled human saliva spiked with inactivated SARS-COV-2 virus gave excellent correlation with viral loads obtained from qPCR (p = 0.0003, r = 0.99).

Bio-modified magnetic nanoparticles with Terminalia arjuna bark extract for the removal of methylene blue and lead (II) from simulated wastewater.

This study reports a greener, cheaper and convenient approach to synthesize Terminalia arjuna bark extract coated magnetite nanoparticles (TA@MNPs) using the co-precipitation method and efficient removal of methylene blue (MB) and lead ions [Pb(II)] from simulated wastewater. The synthesized nanoparticles (NPs) were characterized by various techniques such as DLS, XRD, FTIR, HRTEM, AGM, and TGA. From TGA analysis, TA@MNPs was found to be stable even after 500 °C. Using the batch method, maximum removal was achieved at pH 9.0 for MB and pH 3.0 for Pb(II) solutions, respectively. Adsorption study showed that TA@MNPs followed pseudo-second-order kinetics by both adsorbates while isotherm modeling towards adsorption of Pb(II) and MB exhibited Langmuir and Freundlich isotherm respectively. The maximum adsorption capacity for Pb(II) on TA@MNPs was 210.5 mg g-1. The thermodynamic study proved the spontaneity of the physisorption process. Regeneration studies were also performed using five different eluents for the two adsorbents. Overall, TA@MNPs effectively removed pollutants from wastewater and thus could be potentially useful in providing clean water in a cheaper way.

Biomarker imprinted magnetic core-shell nanoparticles for rapid, culture free detection of pathogenic bacteria.

Rapid and selective detection of microorganisms in complex biological systems draws huge attention to address the rising issue of antimicrobial resistance. Diagnostics based on the identification of whole microorganisms are laborious, time-consuming and costly, thus alternative strategies for early clinical diagnosis include biomarker based microbial detection. This paper describes a low-cost, easy-to-use method for the detection of Pseudomonas aeruginosa infections by specifically identifying a biomarker pyocyanin, using surface-molecularly imprinted nanoparticles or "plastibodies". The selective nanopockets are created by templating pyocyanin onto 20 nm allyl-functionalized magnetic nanoparticles coated with a thin layer of the acrylamide-based polymer. This functional material with an impressive imprinting factor (IF) of 5 and a binding capacity of ∼2.5 mg g-1 of polymers can be directly applied for the detection of bacteria in complex biological samples based on the presence of pyocyanin. These MIPs are highly selective and sensitive to pyocyanin and can consistently bind with pyocyanin in repeated use. Finally, the facile and efficient capture of pyocyanin has versatile applications ranging from biomarker based culture free detection of P. aeruginosa to monitoring of the therapeutic regime, in addition to developing a new class of antibiotics.

Magnetic Nanoparticles with Surface Nanopockets for Highly Selective Antibody Isolation.

Monoclonal antibodies (mAbs) are key components of revolutionary disease immunotherapies and are also essential for medical diagnostics and imaging. The impact of cost is illustrated by a price >$200,000 per year per patient for mAb-based cancer therapy. Purification represents a major issue in the final cost of these immunotherapy drugs. Protein A (PrA) resins are widely used to purify antibodies, but resin cost, separation efficiency, reuse, and stability are major issues. This paper explores a synthesis strategy for low-cost, reusable, stable PrA-like nanopockets on core-shell silica-coated magnetic nanoparticles (NPs) for IgG antibody isolation. Mouse IgG2a, a strong PrA binder, was used as a template protein, first attaching it stem-down onto the NP surface. The stem-down orientation of IgG2a on the NP surface before polymerization is critical for designing the films to bind IgGs. Following this, 1-tetraethoxysilane and four organosilane monomers with functional groups capable of mimicking binding interactions of proteins with IgG antibody stems were reacted to form a thin polymer coating on the NPs. After blocking nonspecific binding sites, removal of the mouse IgG2a provided nanopockets on the core-shell NPs that showed binding characteristics for antibodies remarkably similar to PrA. Both smooth and rough core-shell NPs were used, with the latter providing much larger binding capacities for IgGs, with an excellent selectivity slightly better than that of commercial PrA magnetic beads. This paper is the first report of IgG-binding NPs that mimic PrA selectivity. These nanopocket NPs can be used for at least 15 regeneration cycles, and cost/use was 57-fold less than a high-quality commercial PrA resin.

Novel epoxy-silica nanoparticles to develop non-enzymatic colorimetric probe for analytical immuno/bioassays.

We have developed a novel method to develop epoxy silica nanoparticles (EfSiNP) in a single pot. High surface coverage of epoxy functional groups between 150 and 57000 molecules per particles (∼1013-1016 molecules/mL of 200 nm EfSiNPs) was achieved for different preparation conditions. We then created a red colored probe by conjugating Fuchsin dye to the epoxy functionalities of EfSINPs. Anti-mouse IgG was co-immobilized with Fuchsin and their ratios were optimized for achieving optimum ratios by testing those in functional assays. Dye to antibody ratios were in good negative correlation with a coefficient of -1.00 measured at a confidence level of over 99%. We employed the developed non-enzymatic colorimetric immunonanoprobe for detecting mouse IgG in a direct immunoassay format. We achieved a sensitivity of 427 pg/mL with the assay.

Automated 3D-Printed Microfluidic Array for Rapid Nanomaterial-Enhanced Detection of Multiple Proteins.

We report here the fabrication and validation of a novel 3D-printed, automated immunoarray to detect multiple proteins with ultralow detection limits. This low cost, miniature immunoarray employs electrochemiluminescent (ECL) detection measured with a CCD camera and employs touch-screen control of a micropump to facilitate automated use. The miniaturized array features prefilled reservoirs to deliver sample and reagents to a paper-thin pyrolytic graphite microwell detection chip to complete sandwich immunoassays. The detection chip achieves high sensitivity by using single-wall carbon nanotube-antibody conjugates in the microwells and employing massively labeled antibody-decorated RuBPY-silica nanoparticles to generate ECL. The total cost of an array is $0.65, and an eight-protein assay can be done in duplicate for $0.14 per protein with limits of detection (LOD) as low as 78-110 fg mL-1 in diluted serum. The electronic control system costs $210 in components. Utility of the automated immunoarray was demonstrated by detecting an eight-protein prostate cancer biomarker panel in human serum samples in 25 min. The system is well suited to future clinical and point-of-care diagnostic testing and could be used in resource-limited environments.

Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array.

Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in SN2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu2+ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2'-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 106 nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD50 and Comet assay results.

Albumin removal from human serum using surface nanopockets on silica-coated magnetic nanoparticles.

Selective removal of albumin from human serum is an essential step prior to proteomic analyses, especially when using mass spectrometry. Here we report stable synthetic nanopockets on magnetic nanoparticle surfaces that bind to human serum albumin (HSA) with high affinity and specificity. The nanopockets are created by templating HSA on 200 nm silica-coated paramagnetic nanoparticles using polymer layers made using 4 organo-silane monomers. These monomers have amino acid-like side chains providing hydrophobic, hydrophilic and H-bonding interactions that closely mimic features of binding sites on antibodies. The binding capacity of the material was 21 mg HSA g-1, and consistently removed ∼88% albumin from human serum in multiple repeated use.

Fe3O4 nanoparticles on graphene oxide sheets for isolation and ultrasensitive amperometric detection of cancer biomarker proteins.

Ultrasensitive mediator-free electrochemical detection for biomarker proteins was achieved at low cost using a novel composite of Fe3O4 nanoparticles loaded onto graphene oxide (GO) nano-sheets (Fe3O4@GO). This paramagnetic Fe3O4@GO composite (1µm size range) was decorated with antibodies against prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA), and then used to first capture these biomarkers and then deliver them to an 8-sensor detection chamber of a microfluidic immunoarray. Screen-printed carbon sensors coated with electrochemically reduced graphene oxide (ERGO) and a second set of antibodies selectively capture the biomarker-laden Fe3O4@GO particles, which subsequently catalyze hydrogen peroxide reduction to detect PSA and PSMA. Accuracy was confirmed by good correlation between patient serum assays and enzyme-linked immuno-sorbent assays (ELISA). Excellent detection limits (LOD) of 15 fg/mL for PSA and 4.8 fg/mL for PSMA were achieved in serum. The LOD for PSA was 1000-fold better than the only previous report of PSA detection using Fe3O4. Dynamic ranges were easily tunable for concentration ranges encountered in serum samples by adjusting the Fe3O4@GO Concentration. Reagent cost was only $0.85 for a single 2-protein assay.

Fast nucleation for silica nanoparticle synthesis using a sol-gel method.

We have developed a method that for the first time allowed us to synthesize silica particles in 20 minutes using a sol-gel preparation. Therefore, it is critically important to understand the synthesis mechanism and kinetic behavior in order to achieve a higher degree of fine tuning ability during the synthesis. In this study, we have employed our ability to modulate the physical nature of the reaction medium from sol-gel to emulsion, which has allowed us to halt the reaction at a particular time; this has allowed us to precisely understand the mechanism and chemistry of the silica polymerization. The synthesis medium is kept quite simple with tetraethyl orthosilicate (TEOS) as a precursor in an equi-volumetric ethanol-water system and with sodium hydroxide as a catalyst. Synthesis is performed under ambient conditions at 20 °C for 20 minutes followed by phasing out of any unreacted TEOS and polysilicic acid chains via their emulsification with supersaturated water. We have also demonstrated that the developed particles with various sizes can be used as seeds for further particle growth and other applications. Luminol, a chemiluminescent molecule, has been entrapped successfully between the layers of silica and was demonstrated for the chemiluminescence of these particles.

Sodium hydroxide catalyzed monodispersed high surface area silica nanoparticles.

Understanding of the synthesis kinetics and our ability to modulate medium conditions allowed us to generate nanoparticles via an ultra-fast process. The synthesis medium is kept quite simple with tetraethyl orthosilicate (TEOS) as precursor and 50% ethanol and sodium hydroxide catalyst. Synthesis is performed under gentle conditions at 20 °C for 20 min Long synthesis time and catalyst-associated drawbacks are most crucial in silica nanoparticle synthesis. We have addressed both these bottlenecks by replacing the conventional Stober catalyst, ammonium hydroxide, with sodium hydroxide. We have reduced the overall synthesis time from 20 to 1/3 h, ~60-fold decrease, and obtained highly monodispersed nanoparticles with 5-fold higher surface area than Stober particles. We have demonstrated that the developed NPs with ~3-fold higher silane can be used as efficient probes for biosensor applications.

Antibody-like Biorecognition Sites for Proteins from Surface Imprinting on Nanoparticles.

Natural antibodies are used widely for important applications such as biomedical analysis, cancer therapy, and directed drug delivery, but they are expensive and may have limited stability. This study describes synthesis of antibody-like binding sites by molecular imprinting on silica nanoparticles (SiNP) using a combination of four organosilane monomers with amino acid-like side chains providing hydrophobic, hydrophilic, and H-bonding interactions with target proteins. This approach provided artificial antibody (AA) nanoparticles with good selectivity and specificity to binding domains on target proteins in a relatively low-cost synthesis. The AAs were made by polymer grafting onto SiNPs for human serum albumin (HSA) and glucose oxidase (GOx). Binding affinity, selectivity, and specificity was compared to several other proteins using adsorption isotherms and surface plasmon resonance (SPR). The Langmuir-Freundlich adsorption model was used to obtain apparent binding constants (KLF) from binding isotherms of HSA (6.7 × 10(4)) and GOx (4.7 × 10(4)) to their respective AAs. These values were 4-300 fold larger compared to a series of nontemplate proteins. SPR binding studies of AAs with proteins attached to a gold surface confirmed good specificity and revealed faster binding for the target proteins compared to nontarget proteins. Target proteins retained their secondary structures upon binding. Binding capacity of AAHSA for HSA was 5.9 mg HSA/g compared to 1.4 mg/g for previously report imprinted silica beads imprinted with poly(aminophenyl)boronic acid. Also, 90% recovery for HSA spiked into 2% calf serum was found for AAHSA.

Resistive-Pulse Measurements with Nanopipettes: Detection of Vascular Endothelial Growth Factor C (VEGF-C) Using Antibody-Decorated Nanoparticles.

Quartz nanopipettes have recently been employed for resistive-pulse sensing of Au nanoparticles (AuNP) and nanoparticles with bound antibodies. The analytical signal in such experiments is the change in ionic current caused by the nanoparticle translocation through the pipette orifice. This paper describes resistive-pulse detection of cancer biomarker (Vascular Endothelial Growth Factor-C, VEGF-C) through the use of antibody-modified AuNPs and nanopipettes. The main challenge was to differentiate between AuNPs with attached antibodies for VEGF-C and antigen-conjugated particles. The zeta-potentials of these types of particles are not very different, and, therefore, carefully chosen pipettes with well-characterized geometry were necessary for selective detection of VEGF-C.

3D-Printed Fluidic Devices for Nanoparticle Preparation and Flow-Injection Amperometry Using Integrated Prussian Blue Nanoparticle-Modified Electrodes.

A consumer-grade fused filament fabrication (FFF) 3D printer was used to construct fluidic devices for nanoparticle preparation and electrochemical sensing. Devices were printed using poly(ethylene terephthalate) and featured threaded ports to connect polyetheretherketone (PEEK) tubing via printed fittings prepared from acrylonitrile butadiene styrene (ABS). These devices included channels designed to have 800 µm × 800 µm square cross sections and were semitransparent to allow visualization of the solution-filled channels. A 3D-printed device with a Y-shaped mixing channel was used to prepare Prussian blue nanoparticles (PBNPs) under flow rates of 100 to 2000 µL min(-1). PBNPs were then attached to gold electrodes for hydrogen peroxide sensing. 3D-printed devices used for electrochemical measurements featured threaded access ports into which a fitting equipped with reference, counter, and PBNP-modified working electrodes could be inserted. PBNP-modified electrodes enabled amperometric detection of H2O2 in the 3D-printed channel by flow-injection analysis, exhibiting a detection limit of 100 nM and linear response up to 20 µM. These experiments show that a consumer-grade FFF printer can be used to fabricate low-cost fluidic devices for applications similar to those that have been reported with more expensive 3D-printing methods.