A rapid total bacterial count method using gold nanoparticles conjugated with an aptamer for water quality assessment.

Total bacterial count is a routine parameter in microbial safety assessment used in many fields, such as drinking water and industrial water testing. The current gold standard method for counting bacteria is the plate culture method (or heterotrophic plate count) that requires a microbiology laboratory and a long turnover time of at least 24 hours. To tackle these shortcomings, we developed a rapid total bacterial count method that relies on gold nanoparticles (AuNPs) conjugated with affinity ligands to stain bacterial cells captured on a syringe filter. Two affinity ligands were exploited, i.e. a DNA aptamer (AB2) and a lectin Griffonia simplicifolia II (GSII) that recognize bacterial cell wall commonalities, i.e. peptidoglycan and its amino sugars. Upon proper formulation with addition of a surfactant, the AB2 conjugated AuNPs (AB2-AuNPs) can selectively stain bacterial cells captured on the filter membrane with a higher sensitivity than GSII-AuNPs. Measuring the staining intensity using an in-house-built handheld detector allowed us to correlate its intensity reading with the total number of bacterial units present. This bacteria quantification method, referred to as "Filter-and-Stain", had an efficient turnover time of 20 min suggesting its potential usage for rapid on-site applications. Additionally, the detection sensitivity provided by the AB2-AuNP nanoreagent offered a limit of detection as low as 100 CFU mL-1. We have demonstrated the use of the AB2-AuNPs for detection of bacteria from environmental water samples.

A portable SERS sensor for pyocyanin detection in simulated wound fluid and through swab sampling.

A portable surface-enhanced Raman spectroscopy (SERS) sensor for detecting pyocyanin (PYO) in simulated wound fluid and from bacteria samples was developed. Solution-phase SERS detection protocols are designed to be compatible with two different clinical practices for wound exudate collection, namely negative pressure liquid collection and swabbing. For citrate-coated metal nanoparticles of three different compositions, i.e. gold (AuNPs), alloyed silver/gold (AgAuNPs), and silver (AgNPs), we firstly confirmed their interaction with PYO in the complex wound fluid, using fluorescence quenching experiments, which rationalized the Raman enhancement effects. We then demonstrated the Raman enhancement effects of the metal nanoparticles in the order of AgNPs > AgAuNPs > AuNPs. The limit of detection (LOD) achieved for PYO is 1.1 µM (in a linear range of 0.1-25 µM by the AgNPs), 10.9 µM (in a linear range of 5-100 µM, by the AgAuNPs), and 17.7 µM (in a linear range of 10-100 µM by the AuNPs). The AgNP and AgAuNP sensors together cover the sensitivity and dynamic range requirements for the clinical detection of wound infection, where PYO is present at a concentration of 1-50 µM. In addition, sterilized cotton swabs were used to collect wound fluid and transfer samples into AgNP solution for SERS measurements. This detection protocol was completed within 5 minutes with a LOD of 23.1 µM (in a linear range of 15-100 µM). The SERS sensing protocol was validated by its successful detection of PYO in cultured Pseudomonas aeruginosa bacteria. The findings presented in this work pave the way towards point-of-care diagnostics of wound infections.

Epitope-Functionalized Gold Nanoparticles for Rapid and Selective Detection of SARS-CoV-2 IgG Antibodies.

Rapid and inexpensive immunodiagnostic assays to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroconversion are essential for conducting large-scale COVID-19 epidemiological surveillance and profiling humoral responses against SARS-CoV-2 infections or immunizations. Herein, a colorimetic serological assay to detect SARS-CoV-2 IgGs in patients' plasma was developed using short antigenic epitopes conjugated to gold nanoparticles (AuNPs). Four immunodominant linear B-cell epitopes, located on the spike (S) and nucleocapsid (N) proteins of SARS-CoV-2, were characterized for their IgG binding affinity and used as highly specific biological motifs on the nanoparticle to recognize target antibodies. Specific bivalent binding between SARS-CoV-2 antibodies and epitope-functionalized AuNPs trigger nanoparticle aggregation, which manifests as a distinct optical transition in the AuNPs' plasmon characteristics within 30 min of antibody introduction. Co-immobilization of two epitopes improved the assay sensitivity relative to single-epitope AuNPs with a limit of detection of 3.2 nM, commensurate with IgG levels in convalescent COVID-19-infected patients. A passivation strategy was further pursued to preserve the sensing response in human plasma medium. When tested against 35 clinical plasma samples of varying illness severity, the optimized nanosensor assay can successfully identify SARS-CoV-2 infection with 100% specificity and 83% sensitivity. As the epitopes are conserved within the circulating COVID-19 variants, the proposed platform holds great potential to serve as a cost-effective and highly specific alternative to classical immunoassays employing recombinant viral proteins. These epitope-enabled nanosensors further expand the serodiagnostic toolbox for COVID-19 epidemiological study, humoral response monitoring, or vaccine efficiency assessment.

Effect of natural biopolymers on amyloid fibril formation and morphology.

Amyloid fibrils are associated with the pathogenesis of protein misfolding diseases such as Alzheimer's disease. These fibrils typically exhibit different morphologies when grown in vitro, and this has been known to affect their biological properties and cytotoxicity. The formation kinetics and resultant morphology of fibrils formed from the model proteins Bovine Insulin and Hen Egg White Lysozyme have been measured. We show that the presence of gum arabic and pectin during fibril formation cause the amyloid fibrils formed to associate into higher order fibrillar aggregates. It is postulated that the carbohydrates act as a template to promote inter-fibril association, resulting in larger, thicker fibrils. This observation provides some insight into the differences in growing amyloid fibrils in vitro in the absence or presence of other high molecular weight compounds. Furthermore, these findings suggest a method of tailoring fibril structure for applications in nanotechnology and bio-template applications.

Nanomaterials-based biosensors for detection of microorganisms and microbial toxins.

Detection of microorganisms and microbial toxins is important for health and safety. Due to their unique physical and chemical properties, nanomaterials have been extensively used to develop biosensors for rapid detection of microorganisms with microbial cells and toxins as target analytes. In this paper, the design principles of nanomaterials-based biosensors for four selected analyte categories (bacteria cells, toxins, mycotoxins, and protozoa cells), closely associated with the target analytes' properties is reviewed. Five signal transducing methods that are less equipment intensive (colorimetric, fluorimetric, surface enhanced Raman scattering, electrochemical, and magnetic relaxometry methods) is described and compared for their sensory performance (in term oflimit of detection, dynamic range, and response time) for all analyte categories. In the end, the suitability of these five sensing principles for on-site or field applications is discussed. With a comprehensive coverage of nanomaterials, design principles, sensing principles, and assessment on the sensory performance and suitability for on-site application, this review offers valuable insight and perspective for designing suitable nanomaterials-based microorganism biosensors for a given application.

A generic class of amyloid fibril inhibitors.

Amyloid fibrils are large ordered fibrillar aggregates formed from mis-folded proteins. A number of human diseases are linked to the presence of amyloid deposits, including Alzheimer's disease, Parkinson's disease and type II diabetes. One therapeutic strategy for treating amyloid related diseases involves inhibiting fibril formation. Amyloid fibrils are ß-sheet rich fibrillar aggregates that associate through hydrophobic interactions between precursor units. In this study, these generic physical properties of amyloid fibrils have been exploited to design a universal class of amphiphilic macromolecular inhibitors. A naturally occurring macromolecule of this structure is arabinogalactan protein (AGP), a component of gum arabic (GA). In addition, two synthetic polymers based on the proposed amphiphilic structure were synthesized and tested. These synthetic mimics, referred to as poly(norbornene glucose ester) (PNGE) and poly(norbornene gluconamide) (PNGA), possess hydrophobic polynorbornene backbones and pendent hydrophilic cyclic and open-chain glucose units, respectively. AGP, PNGE and PNGA all show inhibitory effects on in vitro amyloid fibril formation in bovine insulin (BI), hen egg white lysozyme (HEWL) and amyloid beta 1-40 (Aß) proteins. Circular dichroism (CD) spectra of the proteins in the presence of the inhibitors suggests that amyloid fibril formation is inhibited by stabilization of the native α-helices of the proteins, as well as binding of the inhibitors to the ß-sheet precursors. Based upon these results, glycosylated hydrophobic macromolecules are identified as a promising class of therapeutic agents for amyloid related diseases. Furthermore, we have determined that the intensity of the fluorescent probe thioflavin T (ThT) is dependent on both fibril morphology and the presence of the inhibitors, and is therefore not a quantitative measure of protein conversion to fibrils.

A brief overview of amyloids and Alzheimer's disease.

Amyloid fibrils are self-assembled fibrous protein aggregates that are associated with a number of presently incurable diseases such as Alzheimer's and Parkinson's disease. Millions of people worldwide suffer from amyloid diseases. This review summarizes the unique cross-ß structure of amyloid fibrils, morphological variations, the kinetics of amyloid fibril formation, and the cytotoxic effects of these fibrils and oligomers. Alzheimer's disease is also explored as an example of an amyloid disease to show the various approaches to treat these amyloid diseases. Finally, this review investigates the nanotechnological and biological applications of amyloid fibrils; as well as a summary of the typical biological pathways involved in the disposal of amyloid fibrils and their precursors.

The effect of concentration, temperature and stirring on hen egg white lysozyme amyloid formation.

Lysozyme is associated with hereditary systemic amyloidosis in humans. Hen egg white lysozyme (HEWL) has been extensively studied as an amyloid forming protein. In this study, we investigated HEWL amyloid formation over a range of temperatures at two stirring speeds and at low concentrations to avoid gel formation. The amyloid fibril formation was found to follow first order kinetics with the rate determining step being the unfolding of the lysozyme. Both the rate of formation and final amount of amyloid formed show maxima with temperature at approximately at 65 °C. CD measurements show that the lysozyme is unfolded by 55 °C. The decrease in amyloid formation at temperatures above 65 °C is attributed to competing amorphous aggregation. The majority of the non-fibrillar aggregates are small and uniform in size with a few larger amorphous aggregates observed in the AFM images.