Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10.

The current study reports on the development of a rapid and cost-effective TB-antigen diagnostic test for the detection of Mycobacterium biomarkers from non-sputum-based samples. Two gold nanoparticle (AuNP)-based rapid diagnostic tests (RDTs) in the form of lateral flow immunoassays (LFIAs) were developed for detection of immunodominant TB antigens, the 6 kDa early secreted antigen target EsxA (ESAT-6) and the 10 kDa culture filtrate protein EsxB (CFP-10). AuNPs were synthesized using the Turkevich method and characterized by UV-vis spectrophotometer and transmission electron microscope (TEM). The AuNP-detection probe conjugation conditions were determined by comparing the stability of 14 nm AuNPs at different pH conditions, following salt challenge. Thereafter, ESAT-6 and CFP-10 antibodies were conjugated to the AuNPs and used for the colorimetric detection of TB antigens. Selection of the best detection and capture antibody pairs was determined by Dot spotting. The limits of detection (LODs) for the LFIAs were evaluated by dry testing. TEM results showed that the 14 nm AuNPs were mostly spherical and well dispersed. The ESAT-6 LFIA prototype had an LOD of 0.0625 ng/mL versus the CFP-10 with an LOD of 7.69 ng/mL. Compared to other studies in the literature, the LOD was either similar or lower, outperforming them. Moreover, in some of the previous studies, an enrichment/extraction step was required to improve on the LOD. In this study, the LFIAs produced results within 15 min and could be suitable for use at PoCs either in clinics, mobile clinics, hospitals or at home by the end user. However, further studies need to be conducted to validate their use in clinical samples.

Synthesis, Characterization, and Electrochemical Evaluation of Copper Sulfide Nanoparticles and Their Application for Non-Enzymatic Glucose Detection in Blood Samples.

Glutathione-capped copper sulfide (CuxSy) nanoparticles with two different average sizes were successfully achieved by using a simple reduction process that involves only changing the reaction temperature. Temperature-induced changes in the size of CuxSy nanoparticles resulted in particles with different optical, morphological, and electrochemical properties. The dependence of electrochemical sensing properties on the sizes of CuxSy nanoparticles was studied by using voltammetric and amperometric techniques. The spherical CuxSy nanoparticles with the average particle size of 25 ± 0.6 nm were found to be highly conductive as compared to CuxSy nanoparticles with the average particle size of 4.5 ± 0.2 nm. The spherical CuxSy nanoparticles exhibited a low bandgap energy (Eg) of 1.87 eV, resulting in superior electrochemical properties and improved electron transfer during glucose detection. The sensor showed a very good electrocatalytic activity toward glucose molecules in the presence of interference species such as uric acid (UA), ascorbic acid (AA), fructose, sodium chloride, and sucrose. These species are often present in low concentrations in the blood. The sensor demonstrated an excellent dynamic linear range between 0.2 to 16 mM, detection limit of 0.2 mM, and sensitivity of 0.013 mA/mM. The applicability of the developed sensor for real field determination of glucose was demonstrated by use of spiked blood samples, which confirmed that the developed sensor had great potential for real analysis of blood glucose levels.

Development of a Versatile Half-Strip Lateral Flow Assay toward the Detection of Rift Valley Fever Virus Antibodies.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease that is caused by the Rift Valley fever virus (RVFV); Bunyaviridae: Phlebovirus. RVF disease can affect several different species, including ruminants, camels and humans and thus present a dual threat to public health and livestock food production in endemic regions. In livestock, the RVFV infection is characterised by an acute hepatitis, abortion and high mortality rates in new-born animals. The current RVF diagnostic techniques have shown good sensitivity. However, they require extensive sample processing and complex instrumentation. Owing to speed, low cost, ease of use, and most importantly, the ability to diagnose diseases at sites where they are managed, lateral flow immunoassays (LFIA) are the most widely used point-of-care (POC) tools for disease diagnosis. In this study, a lateral flow assay (LFA) device that is able to detect antibodies against RVFV, with a minimum detectable concentration of 0.125 mg/mL, was successfully developed. The LFA also successfully detected RVFV antibodies in reference RVFV sera. Protein A (ProA), which has the ability to bind immunoglobulins from different species, was used in the detection probe, giving the developed RVFV LFA potential for multi-species diagnosis.

A Review of the Nucleic Acid-Based Lateral Flow Assay for Detection of Breast Cancer from Circulating Biomarkers at a Point-of-Care in Low Income Countries.

The current levels of breast cancer in African women have contributed to the high mortality rates among them. In South Africa, the incidence of breast cancer is also on the rise due to changes in behavioural and biological risk factors. Such low survival rates can be attributed to the late diagnosis of the disease due to a lack of access and the high costs of the current diagnostic tools. Breast cancer is asymptomatic at early stages, which is the best time to detect it and intervene to prevent high mortality rates. Proper risk assessment, campaigns, and access to adequate healthcare need to be prioritised among patients at an early stage. Early detection of breast cancer can significantly improve the survival rate of breast cancer patients, since therapeutic strategies are more effective at this stage. Early detection of breast cancer can be achieved by developing devices that are simple, sensitive, low-cost, and employed at point-of-care (POC), especially in low-income countries (LICs). Nucleic-acid-based lateral flow assays (NABLFAs) that combine molecular detection with the immunochemical visualisation principles, have recently emerged as tools for disease diagnosis, even for low biomarker concentrations. Detection of circulating genetic biomarkers in non-invasively collected biological fluids with NABLFAs presents an appealing and suitable method for POC testing in resource-limited regions and/or LICs. Diagnosis of breast cancer at an early stage will improve the survival rates of the patients. This review covers the analysis of the current state of NABLFA technologies used in developing countries to reduce the scourge of breast cancer.

Vascular targeted nanotherapeutic approach for obesity treatment.

Obesity is a global epidemic that poses a serious health concern due to it being a risk factor for life-threatening chronic diseases, such as type 2 diabetes, cancer, and cardiovascular diseases. Pharmacotherapy remains the mainstay for the management of obesity; however, its usefulness is limited due to poor drug efficacy, non-specificity and toxic side effects. Therefore, novel approaches that could provide insights into obesity and obesity-associated diseases as well as development of novel anti-obesity treatment modalities or improvement on the existing drugs are necessary. While the ideal treatment of obesity should involve early intervention in susceptible individuals, targeted nanotherapy potentially provides a fresh perspective that might be better than the current conventional therapies. Independent studies have shown improved drug efficacy by using prohibitin (PHB)-targeted therapy in obese rodents and non-human primates, thus providing a proof of concept that targeted nanotherapy can be a feasible treatment for obesity. This review presents a brief global survey of obesity, its impact on human health, its current treatment and their limitations, and the role of angiogenesis and PHB in the development of obesity. Finally, the role and potential use of nanotechnology coupled with targeted drug delivery in the treatment of obesity are discussed.

Peptide-functionalized nanoparticles for the selective induction of apoptosis in target cells.

AIM: The study developed a prohibitin (PHB) targeted nanotherapy for selective induction of apoptosis in target cells. METHODS: Gold nanoparticles (AuNPs) were bifunctionalized with adipose homing and proapoptotic peptides. The efficacy and mode of cell death induced by the AuNPs were investigated in vitro on three cancer cell lines. RESULTS: The antiproliferative activity of PHB-targeted bifunctionalized AuNPs was more pronounced on cells that express the PHB receptor, and demonstrated receptor-mediated targeting and selectivity. The bifunctionalized AuNPs induced cell death by apoptosis. CONCLUSION: The PHB-targeted nanotherapy under study could potentially be used for treatment of diseases that are characterized by overexpression of PHB. As such, further investigations will be conducted in vivo.

Cytotoxicity, intracellular localization and exocytosis of citrate capped and PEG functionalized gold nanoparticles in human hepatocyte and kidney cells.

Surface-modified gold nanoparticles (AuNPs) are nanomaterials that hold promise in drug delivery applications. In this study, the cytotoxicity, uptake, intracellular localization, and the exocytosis of citrate-stabilized (Cit-AuNP) and polyethylene glycol (PEG)-modified gold nanoparticles with the carboxyl (COOH) terminal functional group were assessed in human embryonic kidney (HEK 293) and the human caucasian hepatocytes carcinoma (Hep G2) cell systems, representing two major accumulation sites for AuNPs. The zeta (ζ)-potential measurements confirmed the negative surface charge of the AuNPs in water and in cell growth medium. The transmission electron microscopy confirmed the size and morphology of the AuNPs. Both types of AuNPs were shown to induce cytotoxic effects in cells. The Hep G2 cells were more sensitive cell type, with the COOH-PEG-AuNPs inducing the highest toxicity at higher concentrations. Dark field microscopy and TEM images revealed that the AuNPs were internalized in cells, mostly as agglomerates. TEM micrographs further revealed that the AuNPs were confined as agglomerates inside vesicle-like compartments, likely to be endosomal and lysosomal structures as well as in the cytosol, mostly as individual particles. The AuNPs were shown to remain in cellular compartments for up to 3 weeks, but thereafter, clearance of the gold nanoparticles from the cells by exocytosis was evident. The results presented in this study may therefore give an indication on the fate of AuNPs on long-term exposure to cells and may also assist in safety evaluation of AuNPs.

Facile Attachment of TAT Peptide on Gold Monolayer Protected Clusters: Synthesis and Characterization.

High affinity thiolate-based polymeric capping ligands are known to impart stability onto nanosized gold nanoparticles. Due to the stable gold-sulfur bond, the ligand forms a protective layer around the gold core and subsequently controls the physicochemical properties of the resultant nanogold mononuclear protected clusters (AuMPCs). The choice of ligands to use as surfactants for AuMPCs largely depends on the desired degree of hydrophilicity and biocompatibility of the MPCs, normally dictated by the intended application. Subsequent surface modification of AuMPCs allows further conjugation of additional biomolecules yielding bilayer or multilayered clusters suitable for bioanalytical applications ranging from targeted drug delivery to diagnostics. In this study, we discuss our recent laboratory findings on a simple route for the introduction of Trans-Activator of Transcription (TAT) peptide onto the surface of biotin-derivatised gold MPCs via the biotin-strepavidin interaction. By changing the surface loading of biotin, controlled amounts of TAT could be attached. This bioconjugate system is very attractive as a carrier in intercellular delivery of various delivery cargoes such as antibodies, proteins and oligonucleotides.

Targeted destruction of HIV-positive cells.

INTRODUCTION: HIV/AIDS is now a global epidemic that has become the leading infectious killer of adults worldwide. Although antiretroviral (ARV) therapy has dramatically improved the quality of life and increased the life expectancy of those infected with HIV but frequency of dosing and drug toxicity as well as the development of viral resistance pose additional limitations. The rapidly expanding field of nanotechnology has vast potential to radically advance the treatment and prevention of HIV/AIDS. Nanoparticles can provide improved drug delivery, by virtue of their small size, robustness, safety, multimodality or multifunctionality. AIMS AND OBJECTIVES: Since HIV primarily infects CD4+ cells; we aim to use CD4 as a selectable target to deliver a pro-apoptotic protein to HIV-infected cells using nanoparticles as carriers. The aim of study was to develop a nanotechnology-based death inducing delivery system for the destruction of CD4+HIV infected cells through the activation of caspase-3. METHODOLOGY: A modified caspase-3 protein (Mut-3) was engineered, which is cleavable only by HIV-1 protease. Mut-3 can activate apoptosis in the presence of HIV-1 protease, consequently killing HIV-positive cells. Mut-3 protein was conjugated to gold nanoparticles together with a CD4-targeting peptide. The efficacy of the gold nanoparticles was tested on CHO cells that were genetically engineered to express GFP labelled CD4 and HIV-1 protease. RESULTS: Mut-3 was expressed in bacterial cells and purified. CHO cells that stably over express CD4-GFP and HIV-1 protease were selected using Fluorescence Activated Cell Sorting. Dose response cell culture experiments showed that gold nanoparticles without Mut-3 and CD4-targeting peptide did not induce cell death in CHO cells, while gold nanoparticles that was conjugated with Mut-3 and the CD4-targeting peptide rapidly induced cell death in CHO cells. CONCLUSIONS: Our results suggest that gold nanoparticles conjugated with Mut-3 and a CD4-targeting peptide could potentially induce apoptosis in HIV-infected cells.

Gold nanoparticle based Tuberculosis immunochromatographic assay: the quantitative ESE Quanti analysis of the intensity of test and control lines.

A rapid dual channel lateral flow assay for the detection of Mycobacterium Tuberculosis antibodies (MTB 38 kDa monoclonal antibody) in human blood was developed. The MTB 6-14-38 kDa fusion antigen and anti-Protein A were used as the capture proteins for test and control lines respectively. Protein A labeled 40 nm gold nanoparticles were used as the detection conjugate. Whole blood and serum were spiked with MTB 38 kDa monoclonal antibody to make a positive sample model. The developed lateral flow was used to test MTB 38 kDa monoclonal antibody, and a detection limit of 5 ng/ml was used as a cut-off concentration of the analytes. The effect of the analyte concentration on the MTB lateral flow assay was studied using the variation of the intensity obtained from a ESE Quanti reader. There was a direct correlation between the analyte (MTB 38 kDa monoclonal antibody) concentration and the intensity of the test line. The intensity increased with an increase in the concentration of MTB 38 kDa monoclonal antibody, while in contrast, an increase in analyte concentration decreased the intensity of the control line.

Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines.

BACKGROUND: Reliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles. In this study, the cytotoxicity and uptake of 14 nm and 20 nm citrate stabilised gold nanoparticles (AuNPs) in the bronchial epithelial cell line BEAS-2B, the Chinese hamster ovary cell line CHO, and the human embryonic kidney cell line HEK 293 were investigated. METHODS: Cytotoxicity of the AuNPs was assessed via traditional XTT-, LDH-, and ATP-based assays, followed by cell impedance studies. Dark-field imaging and hyperspectral imaging were used to confirm the uptake of AuNPs into the cells. RESULTS: Interference of the AuNPs with the XTT- and ATP-based assays was overcome through the use of cell impedance technology. AuNPs were shown to be relatively non-toxic using this methodology; nevertheless CHO cells were the most sensitive cell type with 20 nm AuNPs having the highest toxicity. Uptake of both 14 nm and 20 nm AuNPs was observed in all cell lines in a time- and cell type-dependent manner. CONCLUSIONS: Using the cell impedance and dark-field hyperspectral imaging technologies, it was possible to study the toxicity of AuNPs in different cell lines and show that these cells could internalize AuNPs with their subsequent intracellular aggregation. It was also possible to show that this toxicity would not correlate with the level of uptake but it would correlate with cell-type and the size of the AuNPs. Therefore, these two label-free methodologies used in this study are suitable for in vitro studies on the effects of AuNPs, and could present themselves as appropriate and valuable methodologies for future nanoparticle toxicity and uptake studies.

De-regulation of the RBBP6 isoform 3/DWNN in human cancers.

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. The human RBBP6 gene codes for three protein isoforms and isoform 3 consists of the domain with no name domain only whilst the other two isoforms, 1 and 2 comprise of additional zinc, RING, retinoblastoma and p53 binding domains. In this study, the localization of RBBP6 using RBBP6 variant 3 mRNA-specific probe was performed to investigate the expression levels of the gene in different tumours and find a link between RBBP6 and human carcinogenesis. Using FISH, real-time PCR and Western blotting analysis our results show that RBBP6 isoform 3 is down-regulated in human cancers. RBBP6 isoform 3 knock-down resulted in reduced G2/M cell cycle arrest whilst its over-expression resulted in increased G2/M cell cycle arrest using propidium iodide DNA staining. The results further demonstrate that the RBBP6 isoform 3 may be the cell cycle regulator and involved in mitotic apoptosis not the isoform 1 as previously reported for mice. In conclusion, these findings suggest that RBBP6 isoform 3 is a cell cycle regulator and may be de-regulated in carcinogenesis.

RBBP6 interacts with multifunctional protein YB-1 through its RING finger domain, leading to ubiquitination and proteosomal degradation of YB-1.

RBBP6 (retinoblastoma binding protein 6) is a 250-kDa multifunctional protein that interacts with both p53 and pRb and has been implicated in mRNA processing. It has also been identified as a putative E3 ubiquitin ligase due to the presence of a RING finger domain, although no substrate has been identified up to now. Using the RING finger domain as bait in a yeast two-hybrid screen, we identified YB-1 (Y-box binding protein 1) as a binding partner of RBBP6, localising the interaction to the last 62 residues of YB-1. We showed, furthermore, that both full-length RBBP6 and the isolated RING finger domain were able to ubiquitinate YB-1, resulting in its degradation in the proteosome. As a result, RBBP6 was able to suppress the levels of YB-1 in vivo and to reduce its transactivational ability. In the light of the important role that YB-1 appears to play in tumourigenesis, our results suggest that RBBP6 may be a relevant target for therapeutic drugs aimed at modifying the activity of YB-1.