Automatic diagnosis of tuberculosis disease based on plasmonic ELISA and color-based image classification

Tuberculosis (TB) remains one of the most devastating infectious diseases and its treatment efficiency is majorly influenced by the stage at which infection with the TB bacterium is diagnosed. The available methods for TB diagnosis are either time consuming, costly or not efficient. This study employs a signal generation mechanism for biosensing, known as Plasmonic ELISA, and computational intelligence to facilitate automatic diagnosis of TB. Plasmonic ELISA enables the detection of a few molecules of analyte by the incorporation of smart nanomaterials for better sensitivity of the developed detection system. The computational system uses k-means clustering and thresholding for image segmentation. This paper presents the results of the classification performance of the Plasmonic ELISA imaging data by using various types of classifiers. The five-fold cross-validation results show high accuracy rate (>97%) in classifying TB images using the entire data set. Future work will focus on developing an intelligent mobile-enabled expert system to diagnose TB in real-time. The intelligent system will be clinically validated and tested in collaboration with healthcare providers in Malaysia

Development of fluorescence based DNA biosensor utilizing quantum dot for DNA of Ganoderma boninense

In this work, fluorescence based DNA biosensor has been developed for detection of ganoderma boninense utilizing quantum dot as sensing material. The DNA sensor was fabricated by modification of hydrophobic surface of CdSe/ZnS quantum dot (CdSe/ZnS QD) nanoparticle with mercaptopropionic acid (MPA) to form water-soluble CdSe/ZnS QD. The modified CdSe/ZnS QD was successfully attached to the ssDNA to form CdSe/ZnS QD-ssDNA conjugate. Attachment of CdSe/ZnS QD with ssDNA and hybridization procedures are divided into procedure 1 and 2. Hybridization of complementary target DNA with CdSe/ZnS QD-ssDNA conjugate and reporter probe labeled with Cy5 is monitored using fluorescence resonance energy transferred (FRET) as detection mode. The FRET of CdSe/ZnS QD-dsDNA is distinguishable from CdSe/ZnS QD-ssDNA conjugate based on no FRET was observed without target DNA system. Several parameters were studied to determine the optimum conditions of hybridization efficiency including hybridization time and hybridization temperature. The highest FRET intensity was observed at 10 minutes hybridization with hybridization temperature of 45 °C for procedure 1 and 25 °C for procedure 2. For sensitivity study, decreased of FRET intensity was observed when the concentration of target DNA increased. Limit of detection (LOD) for procedure 1 is 2.4x10-13 M and procedure 2 is 1.12x10-12 M. Selectivity study for the developed system confirmed that non-complementary DNA shows no FRET signal but only emission of QD. The hybridization did not occur due to QD was not in position with reporter probe for FRET process to occur. This proved that the detection system specific towards target DNA. Reproducibility shows acceptable relative standard deviation (R.S.D) of 1.67 % (n=5) and 0.86 % (n=5) for procedure 1 and 2 respectively. In TEM characterization, the particle size of CdSe/ZnS QD and CdSe/ZnS QD-ssDNA conjugate is within the range of 2 to 10 nm. Agglomeration was observed for CdSe/ZnS QD-ssDNA conjugate

Electrochemical and optical-based immunosensor for detection of Mycobacterium tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a major obstacle for the global that effect the rate of morbidity and mortality. Many attempts and affords had been taken such as developing the drugs for controlling TB, curing the patients and preventing further transmission of the disease. Conventional diagnosis tool of TB often time-consuming, required loads of samples, less sensitive, impractical and costly for point of care diagnostic. In this research, a novel for diagnosis of TB was developed by an optical and electrochemical immunosensor via antibody-antigen interaction for the detection of TB-protein biomarker CFP10-ESAT6. For optical immunosensor, we studied a naked eye detection for TB by utilizing plasmonic enzyme-linked immunosorbent assay (ELISA) for the detection of protein biomarker, Mycobacterium tuberculosis ESAT-6-like protein esxB (CFP10-ESAT6). Here, the biocatalytic cycle of the intracellular enzymes,catalase links to the formation and successive growth of the gold nanoparticles (AuNPs). The formation of blue and red of AuNPs colored solutions links directly to the absence or presence of the TB analytes in the sample solutions. The immunoassay involves catalase-labeled antibodies which consume hydrogen peroxide for reduction of gold (III) chloride and further produce AuNPs. The fast rate of reaction determines the agglomerated of AuNPs for blue solutions while slow reaction for red solution which from monodispersed AuNPs. This serves as a confirmation for the naked eye detection of TB analytes. The optimum concentration of H2O2 and gold ion were 150 µM and 0.25 mM respectively. In the presence of CFP10-ESAT6, blue color produced while in the absence of CFP10-ESAT6, red solution appeared. The detection limit (LOD) of- developed method was 0.01 µg/mL by the naked eye. Additionally, the plasmonic ELISA shows high specificity towards CFP10-ESAT6 protein compared with MPT64 and BSA. Furthermore, our developed technique was successfully tested and confirmed with sputum samples from patients diagnosed with positive and negative TB with good reproducibility. The results show only positive TB sputum samples produced blue color solutions compared with negative sputum samples, non-tuberculosis Runyon Group IV and Mycobacterium fortuitum chelonae complex. The results provided enough evidence for the utilization of our technique in the early diagnosis of TB disease. For electrochemical immunosensor, a modified quantum dot with thioglycolic acid (TGA) (CdSe-ZnS QD) and functionalized silica nanoparticles (SiO2NPs) as modifiers were prepared to enhance performance of disposable screen printed carbon electrode (SPCE). CdSe-ZnS QD was characterized by using fluorescence spectroscopy and High Resolution Transmission Electron Microscopy (HRTEM) while SiNPs with Transmission Electron Microscopy (TEM) and Fourier Transform Infrared (FTIR). Functionalized SiO2NPs and CdSe-ZnS QD were dropped cast on the working electrode for preparation of CdSe-ZnS QD/ SiO2NPs/SPCE. Electrochemical studies using cyclic voltammetry (CV) performed with SiO2NPs/SPCE and CdSe-ZnS QD/SiO2NPs/SPCE were found to give a better response through the optimization of numerous analytical parameters. The modified SPCE was characterized using Field Emission Scanning Electron Microscope (FESEM) and Energy Dispersive X-ray (EDX) respectively. The CdSe-ZnS QD/SiO2NPs/SPCE modified electrode showed increment of active surface area with 4.14 folds higher than bare SPCE. Then indirect ELISA immunoassay was performed on the modified electrode for CFP10-ESAT6 detection using differential potential voltammetry (DPV). DPV current response increased in the presence of CFP10-ESAT6 while decreased in the absence of CFP10-ESAT6. Other than that, DPV current was high for CFP10-ESAT6 compared with BSA and MPT64. The detection of CFP10-ESAT6 showed a linear response towards 2 different concentration of CFP10-ESAT6 with R = 0.9487 for calibration curve. The detection limit of 3.3 x 10-11 g/mL was achieved for linear range of 20 to 100 ng/mL of CFP10-ESAT6 concentration. The proposed methods showed good selectivity and reproducibility of target analyte with RSD value of 1.45 %. As summary, the developed optical immunosensor utilized plasmonic ELISA marked as suitable quantitative method for naked eye detection of TB. Besides, the developed electrochemical immunosensor which used the fabricated electrode can be used as qualitative technique for TB

Physical, Mechanical and Perforation Resistance of Natural-Synthetic Fiber Interply Laminate Hybrid Composites

Natural and synthetic fibres have emerged in high demand due to their excellent properties. Natural fibres have good mechanical properties and are less expensive, making them a viable substitute for synthetic fibers. Owing to certain drawbacks such as their inconsistent quality and hydrophilic nature, researchers focused on incorporating these two fibres as an alternative to improve the limitations of the single fibre. This review focused on the interply hybridisation of natural and synthetic fibres into composites. Natural fibres and their classifications are discussed. The physical and mechanical properties of these hybrid composites have also been included. A full discussion of the mechanical properties of natural/synthetic fibre hybrid composites such as tensile, flexural, impact, and perforation resistance, as well as their failure modes, is highlighted. Furthermore, the applications and future directions of hybrid composites have been described in details

Mechanical Properties of PALF/Kevlar-Reinforced Unsaturated Polyester Hybrid Composite Laminates

Natural and synthetic fibres are in high demand due to their superior properties. Natural fibres are less expensive and lighter as compared to synthetic fibres. Synthetic fibres have drawn much attention, especially for their outstanding properties, such as durability, and stability. The hybridisation between natural and synthetic fibres composite are considered as an alternative to improve the current properties of natural and synthetic fibres. Therefore, this study aimed to determine the physical and mechanical properties of pineapple leaf fibre (PALF) and Kevlar reinforced unsaturated polyester (UP) hybrid composites. The PALF/Kevlar hybrid composites were fabricated by using hand layup method utilising unsaturated polyester as the matrix. These composites were laid up to various laminated configurations, such as [PKP]s, [PPK]s, [KPP]s, [KKP]s, [PPP]s and [KKK]s, whereby PALF denoted as P and Kevlar denoted as K. Next, they were cut into size and dimensions according to standards. Initially, the density of PALF/Kevlar reinforced unsaturated polyester were evaluated. The highest density result was obtained from [KKK]s, however, the density of hybrid composites was closely indistinguishable. Next, moisture absorption behaviour and its effects on the PALF/Kevlar reinforced unsaturated polyester were investigated. The water absorption studies showed that the hybridisation between all PALF and Kevlar specimens absorbed moisture drastically at the beginning of the moisture absorption test and the percentage of moisture uptake increased with the volume fraction of PALF in the samples. The tensile test indicated that all specimens exhibited nonlinear stress-strain behaviour and shown a pseudo-ductility behaviour. [KKP]s and [KPK]s hybrid composites showed the highest tensile strength and modulus. The flexural test showed that [KPK]s had the highest flexural strength of 164.0 MPa and [KKP]s had the highest flexural modulus of 12.6 GPa. In terms of the impact strength and resistance, [KKP]s outperformed the composite laminates. According to SEM scans, the hybrid composites demonstrated a stronger interfacial adhesion between the fibres and matrix than pure PALF composite

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

Surface enhanced CdSe/ZnS QD/SiNP electrochemical immunosensor for the detection of Mycobacterium tuberculosis by combination of CFP10-ESAT6 for better diagnostic specificity

In this study, an electrochemical immunosensor was introduced for the detection of tuberculosis (TB) via utilization of a modified electrode containing a quantum dot (CdSe/ZnS QD) and functionalized silica nanoparticles (SiNPs) on screen-printed carbon electrode (SPCE) CdSe/ZnS QD/SiNPs/SPCE, by employing indirect enzyme-linked immunosorbent assay (ELISA). Here, the fabricated electrode was linked to the biocatalytic action of enzyme catalase through antigen–antibody binding for the detection of the antigen (CFP10–ESAT6) by means of producing a differential pulse voltammetry (DPV) current. The characterization and cyclic voltammetry (CV) of the modified electrode showed good electrochemical behavior and enhanced high electron transfer between the electrode and analyte. Moreover, the active surface area was 4.14-fold higher than the bare SPCE. The developed method showed high selectivity towards CFP10–ESAT6 compared with the other TB proteins. The detection of CFP10–ESAT6 also showed a linear response towards different concentrations of CFP10–ESAT6 with R2 = 0.9937, yielding a limit of detection (LOD) of as low as 1.5 × 10−10 g/mL for a linear range of 40 to 100 ng/mL of CFP10–ESAT6 concentration. The proposed method showed good reproducibility of target analyte with a relative standard deviation of 1.45%

Central Composite Design for Optimization of Kenaf-Reinforced Epoxy Composite Bonding Performance

Kenaf fiber is gaining prominence because of its ability as a natural-based reinforced material in advanced composites. However, kenaf contains a hygroscopic natural waxy substance that covers the fiber layer, providing a low surface tension and preventing strong bonding with the polymer matrix. The goal of this study is to optimize the blending parameters of kenaf fiber-reinforced epoxy composites by alkali treatment concentration, length, and fiber-matrix loading using central composite design. The maximum tensile strength was obtained at 6.03 wt% of NaOH concentration, fiber loading of 26.02 wt%, and fiber length at 7.39 mm, which showed a strong correlation between experimental and predicted values. The analysis of variance function model indicated that fiber length, sodium hydroxide concentration, and fiber loading all play important roles in mechanical properties of composites. Based on the fracture surface observations, kenaf fiber composite strength was closely related to bonding at fiber-matrix interfaces. The most common failure modes in the samples were voids, matrix fracture, fiber breakage, weak bonding, and fiber pull-out