Development of Corynebacterium glutamicum as staphylococcal-targeting chassis via the construction of autoinducing peptide (AIP)-responsive expression system

Despite increasing reports of antimicrobial activities of commensal and non-pathogenic bacteria such as Corynebacterium spp., previous studies on bioengineered therapeutics traditionally employed probiotics and food-grade bacteria which limits further advancements into microbial therapeutics research. In this study, Corynebacterium glutamicum, a generally recognised as safe (GRAS) and model bacterium was employed as a new chassis for the development of bioengineered corynebacterial chassis tailored towards Staphylococcus sp. via autoinducer peptide (AIP)-based quorum sensing (QS) interactions. To develop C. glutamicum as a staphylococcal-targeting chassis, the bacteria were transformed with the pResponse plasmid harboring AIP-responding accessory regulatory proteins agrAC and red fluorescent protein (RFP) genes under the control of the PaceA and P3 promoter, respectively, which was expected to stimulate the production of fluorescence signals in the presence of AIPs. Fluorescence activity of the C. glutamicum pResponse strain was compared to control C. glutamicum pRFP strain containing only the P3-RFP gene without the agrAC gene cassette. Using AIP-I as the input biomolecule, C. glutamicum pResponse strain fluoresced under different concentrations of AIP-I whereas no fluorescence was observed in the control C. glutamicum pRFP strain. When tested with S. aureus culture supernatant, the pResponse strain exhibited increasing fluorescence over the incubation period with the highest fluorescence signal of 183 relative fluorescence units (R.F.U) was observed at the 48 h point thereby demonstrating a functional QS-responsive protein expression system in bioengineered C. glutamicum. These findings demonstrated the feasibility and promising potential of developing bioengineered C. glutamicum as a staphylococcalresponsive and -targeting chassis

Lateral flow immunoassay for naked eye detection of Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. Detection and control of infectious diseases is a major problem, especially in developing countries. Lateral flow immunoassay (LFIA) has been introduced as a handheld immunoassay-based point-of-care platform for an automated detection of TB. The CFP10-ESAT6 antigen of M. tuberculosis was used as the target in early detection of TB using LFIA strip-based POC strategy. An interesting platform based on optical signals is implemented as a colour change in the detection area that is visible to the naked eye. The gold nanoparticles (AuNPs) were used as the colour probe for the detection of a target of interest. The high-resolution transmission electron microscopy (HRTEM) image and ultraviolet-visible spectrophotometer (UV-Vis) analysis confirmed that the synthesized AuNPs were appropriate for the immunoassay designed. The platform consists of AuNPs conjugated with specific antibodies (Ab) to capture the antigen of M. tuberculosis. Under the capillary effect, sandwich immunoreactions of AuNP-Ab-antigen were performed on the test pad of the immunostrip, which can be observed by the colour signal on the test line of the strip with a short assay time. Furthermore, the newly developed biosensor was utilized in CFP10-ESAT6 antigen detection in human sputum specimens with satisfactory results. The characteristic coloured bands enable visual detection (naked eye) of target analyte without instrumentation. This noninvasive diagnose system which is sputum-based detection could provide user-friendly and affordable diagnostic tests in developing countries

Lateral flow immunoassay for ultrasensitive and affordable naked eye detection of tuberculosis

Lateral flow immunoassays (LFIAs) are advantageous over conventional detection methods in terms of their simplicity and rapidity. These assays have been reported using various types of labels but colloidal gold nanoparticles are still the preferred choice as a label because of their easy synthesis, visual detection and stability. Tuberculosis, or TB, is an infectious bacterial disease caused by Mycobacterium tuberculosis. İt remains one of the deadliest diseases in the world. The detection of Mycobacterium tuberculosis using LFIAs was developed and analyzed using gold nanoparticle

Simultaneous amperometric aptasensor based on diazonium grafted screen-printed carbon electrode for detection of CFP10 and MPT64 biomarkers for early tuberculosis diagnosis

Early diagnosis is highly crucial for life-saving and transmission management of tuberculosis (TB). Despite the low sensitivity and time-consuming issues, TB antigen detection still relies on conventional smear microscopy and culture techniques. To address this limitation, we report the development of the first amperometric dual aptasensor for the simultaneous detection of Mycobacterium tuberculosis secreted antigens CFP10 and MPT64 for better diagnosis and control of TB. The developed sensor was based on the aptamers–antibodies sandwich assay and detected by chronoamperometry through the electrocatalytic reaction between peroxidase-conjugated antibodies, H2O2, and hydroquinone. The CFP10 and MPT64 aptamers were immobilized via carbodiimide covalent chemistry over the disposable dual screen-printed carbon electrodes modified with a 4-carboxyphenyl diazonium salt. Under optimized conditions, the aptasensor achieved a detection limit of 1.68 ng mL−1 and 1.82 ng mL−1 for CFP10 and MPT64 antigens, respectively. The developed assay requires a small sample amount (5 µL) and can be easily performed within 2.5 h. Finally, the dual aptasensor was successfully applied to clinical sputum samples with the obtained diagnostic sensitivity (n = 24) and specificity (n = 13) of 100%, respectively, suggesting the readiness of the developed assay to be used for TB clinical application

Etlingera elatior-mediated synthesis of gold nanoparticles and their application as electrochemical current enhancer

This work presents a simple green synthesis of gold nanoparticles (AuNPs) by using an aqueous extract of Etlingera elatior (torch ginger). The metabolites present in E. elatior, including sugars, proteins, polyphenols, and flavonoids, were known to play important roles in reducing metal ions and supporting the subsequent stability of nanoparticles. The present work aimed to investigate the ability of the E. elatior extract to synthesise AuNPs via the reduction of gold (III) chloride hydrate and characterise the properties of the nanoparticles produced. The antioxidant properties of the E. elatior extract were evaluated by analysing the total phenolic and total flavonoid contents. To ascertain the formation of AuNPs, the synthesised particles were characterised using the ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray (EDX) microscopy, and dynamic light scattering (DLS) measurement. The properties of the green synthesised AuNPs were shown to be comparable to the AuNPs produced using a conventional reducing agent, sodium citrate. The UV-Vis measured the surface plasmon resonance of the AuNPs, and a band centered at 529 nm was obtained. The FTIR results proved that the extract contained the O-H functional group that is responsible for capping the nanoparticles. The HRTEM images showed that the green synthesized AuNPs were of various shapes and the average of the nanoparticles’ hydrodynamic diameter was 31.5 ± 0.5 nm. Meanwhile, the zeta potential of −32.0 ± 0.4 mV indicates the high stability and negative charge of the AuNPs. We further successfully demonstrated that using the green synthesised AuNPs as the nanocomposite to modify the working surface of screen-printed carbon electrode (SPCE/Cs/AuNPs) enhanced the rate of electron transfer and provided a sensitive platform for the detection of Cu(II) ions

Electrochemical immunosensor for detection of Mycobacterium tuberculosis

A rapid and sensitive sandwich electrochemical immunosensor was developed based on the fabrication of the graphene/polyaniline (GP/PANI) nanocomposite onto screen-printed gold electrode (SPGE) for detection of Mycobacterium tuberculosis (M. tuberculosis) antigen. The chemical bonding and morphology of GP/PANI-modified SPGE were studied by Raman spectroscopy and field enhance scanning electron microscopy-energy dispersive X-ray spectroscopy (FESEM-EDX), respectively. From both studies, it clearly showed that GP/PANI was successfully coated onto SPGE through drop cast technique. Cyclic voltammetry (CV) was used to study the electrochemical properties of the modified electrode. The effective surface area for GP/PANI-modified SPGE was enhanced when compared with bare SPGE. Differential pulse voltammetry (DPV) was used to detect the M. tuberculosis antigen. This proposed electrochemical immunosensor is sensitive, low sample volume, rapid, and disposable, which is suitable for tuberculosis detection in real samples

Determination of minimal sequence for zearalenone aptamer by computational docking and application on an indirect competitive electrochemical aptasensor

Aptamers are short single-stranded oligonucleotides (either DNA or RNA) that can fold into well-defined three-dimensional (3D) spatial structures which enable them to capture their specific target by complementary shape interactions. Aptamers are selected from large random libraries through the SELEX process and only a small fraction of the sequence is involved in direct docking with the target. In this paper, we describe the possible truncation variants of zearalenone (ZEA) aptamer which might be an effective binding region for the target. The originally selected zearalenone (ZEA) aptamer was 80-mer in length and shown to bind the target with a high affinity (Kd = 41 ± 5 nM). Herein, computational docking simulation was performed with 15 truncated variants to determine the predicted binding energy and responsible binding site of the aptamer-analyte complex. The results revealed that 5 truncated variants had binding energy lower than - 7.0 kcal/mol. Circular dichroism analysis was performed on the shortlisted aptamer and the conformational change of aptamers was observed with the presence of an analyte. Aptamer Z3IN (29-mer) was chosen as the most enhanced affinity for its target with a dissociation constant of 11.77 ± 1.44 nM. The aptamer was further applied in the electrochemical aptasensor of ZEA based on an indirect competitive format. The results demonstrated that the truncated aptamer leads to an enhancement of the sensitivity of the biosensor

Immuno Nanosensor for the Ultrasensitive Naked Eye Detection of Tuberculosis

In the present study, a beneficial approach for the ultrasensitive and affordable naked eye detection and diagnosis of tuberculosis (TB) by utilizing plasmonic enzyme-linked immunosorbent assay (ELISA) via antibody-antigen interaction was studied. Here, the biocatalytic cycle of the intracellular enzymes links to the formation and successive growth of the gold nanoparticles (GNPs) for ultrasensitive detection. The formation of different colored solutions by the plasmonic nanoparticles in the presence of enzyme labels links directly to the existence or non-existence of the TB analytes in the sample solutions. For disease detection, the adapted protocol is based mainly on the conventional ELISA procedure that involves catalase-labeled antibodies, i.e., the enzymes consume hydrogen peroxide and further produce GNPs with the addition of gold (III) chloride. The amount of hydrogen peroxide remaining in the solution determines whether the GNPs solution is to be formed in the color blue or the color red, as it serves as a confirmation for the naked eye detection of TB analytes. However, the conventional ELISA method only shows tonal colors that need a high concentration of analyte to achieve high confidence levels for naked eye detection. Also, in this research, we proposed the incorporation of protein biomarker, Mycobacterium tuberculosis ESAT-6-like protein esxB (CFP-10), as a means of TB detection using plasmonic ELISA. With the use of this technique, the CFP-10 detection limit can be lowered to 0.01 µg/mL by the naked eye. Further, our developed technique was successfully tested and confirmed with sputum samples from patients diagnosed with positive TB, thereby providing enough evidence for the utilization of our technique in the early diagnosis of TB disease

Recent advances in single-cell engineered live biotherapeutic products research for skin repair and disease treatment

Abstract The human microbiome has emerged as a key player in maintaining skin health, and dysbiosis has been linked to various skin disorders. Amidst growing concerns regarding the side effects of antibiotic treatments, the potential of live biotherapeutic products (LBPs) in restoring a healthy microbiome has garnered significant attention. This review aims to evaluate the current state of the art of the genetically or metabolically engineered LBPs, termed single-cell engineered LBPs (eLBPs), for skin repair and disease treatment. While some studies demonstrate promising outcomes, the translation of eLBPs into clinical applications remains a significant hurdle. Substantial concerns arise regarding the practical implementation and scalability of eLBPs, despite the evident potential they hold in targeting specific cells and delivering therapeutic agents. This review underscores the need for further research, robust clinical trials, and the exploration of current advances in eLBP-based bioengineered bacterial chassis and new outlooks to substantiate the viability and effectiveness of eLBPs as a transformative approach in skin repair and disease intervention