Araz Norouz Dizaji

Oxygen levels in the body/and organs significantly influence the regulation of metabolic reactions. Hypoxia, a decrease in oxygen levels, can potentially trigger various signals leading to serious health issues. This study aimed to develop an electrochemical immunosensor platform for rapidly and accurately detecting the hypoxia biomarker, HIF-1α protein. In this regard, screen-printed gold electrodes were modified using 11-mercaptoundecanoic acid and 3-mercapto-1-propanol (as a spacer) to generate functional carboxyl groups. Employing EDC-NHS chemistry facilitated the immobilization of HIF-1α antibodies, which were then utilized for the selective and specific recognition of their target. Electrochemical voltametric measurements were conducted using a potassium ferri/ferrocyanide redox couple in both hypoxia-cultured cell lysates and phosphate-buffered saline spiked with HIF-1α protein.In addition to electrochemical measurements, Western blotting (WB) was performed to compare findings with electrochemical results and to confirm the presence of HIF-1α in hypoxic cell lysates. While WB results only exhibited the qualitative presence of the respective antigen in lysates, significant signal decreases were observed in both Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) measurements due to specific antibody-target binding, emphasizing the electrochemical sensor’s performance for more rapid and quantitative protein detection. The low limit of detection (1.46 nM) suggests the potential of our proposed immunosensor platform for detecting HIF-1α protein within a clinically significant range, which is highly desired for point-of-care applications. This study is one of its kind in the literature to develop an electrochemical immunosensor platform for the rapid detection of HIF-1α. The sensor's ability to provide inexpensive, rapid, and quantitative measurements is a significant advancement for HIF-1 α detection.

One quarter of the global population is thought to be latently infected by Mycobacterium tuberculosis (TB) with it estimated that 1 in 10 of those people will go on to develop active disease. Due to the fact that M. tuberculosis (TB) is a disease most often associated with low‐ and middle‐income countries, it is critical that low‐cost and easy‐to‐use technological solutions are developed, which can have a direct impact on diagnosis and prescribing practice for TB. One area where intervention could be particularly useful is antibiotic susceptibility testing (AST). This work presents a low‐cost, simple‐to‐use AST sensor that can detect drug susceptibility on the basis of changing RNA abundance for the typically slow‐growing M. tuberculosis (TB) pathogen in 96 h using screen‐printed electrodes and standard molecular biology laboratory reactionware. In order to find out the sensitivity of applied sensor platform, a different concentration (10 ⁸ –10 ³ CFU/mL) of M. tuberculosis was performed, and limit of detection and limit of quantitation were calculated as 10 ^3.82 and 10 ^11.59 CFU/mL, respectively. The results display that it was possible to detect TB sequences and distinguish antibiotic‐treated cells from untreated cells with a label‐free molecular detection. These findings pave the way for the development of a comprehensive, low‐cost, and simple‐to‐use AST system for prescribing in TB and multidrug‐resistant tuberculosis.

Accurate diagnosis of cancer cells in early stages plays an important role in reliable therapeutic strategies. In this study, we aimed to develop fluorescence-conjugated polymer carrying nanocapsules (NCs) which is highly selective for myeloma cancer cells. To gain specific targeting properties, NCs, XT5 molecules (a benzamide derivative) which shows high affinity properties against protease-activated receptor-1 (PAR1), that overexpressed in myeloma cancer cells, was used. For this purpose, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000]-carboxylic acid (DSPE-PEG ₂₀₀₀ -COOH) molecules, as a main encapsulation material, was conjugated to XT5 molecules due to esterification reaction using N,N′-dicyclohexylcarbodiimide as a coupling agent. The synthesized DSPE-PEG ₂₀₀₀ -COO-XT5 was characterized by using FT-IR and ¹ H NMR spectroscopies and results indicated that XT5 molecules were successfully conjugated to DSPE-PEG ₂₀₀₀ -COOH. Poly(fluorene-alt-benzothiadiazole) (PFBT) conjugated polymer (CP) was encapsulated with DSPE-PEG ₂₀₀₀ -COO-XT5 due to dissolving in tetrahydrofuran and ultra-sonication in an aqueous solution, respectively. The morphological properties, UV–vis absorbance, and emission properties of obtained CP encapsulated D SPE- P EG ₂₀₀₀ −COO- XT5 (CPDP-XT5) NCs was determined by utilizing scanning electron microscopy, UV–vis spectroscopy, and fluorescent spectroscopy, respectively. Cytotoxicity properties of CPDP-XT5 was evaluated by performing MTT assay on RPMI 8226 myeloma cell lines. Cell viability results confirmed that XT5 molecules were successfully conjugated to DSPE-PEG ₂₀₀₀ -COOH. Specific targeting properties of CPDP-XT5 NCs and XT5-free NCs (CPDP NCs) were investigated on RPMI 8226 myeloma cell lines by utilizing fluorescent microscopy and results indicated that CPDP-XT5 NCs shows significantly high affinity in comparison to CPDP NCs against the cells. Homology modeling and molecular docking properties of XT5 molecules were evaluated and simulation results confirmed our results.

Electrochemical impedance spectroscopy is often used for biomolecular detection based on the interaction of a molecule with a receptor functionalised electrode surface and consequent impedance change. Though its performance is well established, there is still a need for improved sensitivity and specificity, especially when attempting to detect nucleic acids from clinical samples with minimal amplification steps. Localised heating is a potential approach for improving nucleic hybridisation rates and reducing non-specific interactions, and thereby producing high sensitivity and selectivity. The aim of the study was therefore to develop a microheater surrounding Au thin film electrodes, an integrated hybrid chip, for detecting genes of Mycobacterium tuberculosis with enhanced sensitivity. The performance of the integrated hybrid chip was determined using the changes in the charge transfer resistance ( R _ct ) upon DNA hybridisation using probe sequences for M. tuberculosis . Heat transfer within the system was simulated by using COMSOL Multiphysics as a mathematical modelling tool. When a temperature of 50 °C was applied to the microheater during DNA hybridisation steps, R _ct values (which were indicative of DNA–DNA hybridisation) increased 236% and 90% as opposed to off-chip non-heated experiments and off-chip heated experiments. It is concluded from these observations that the microheater indeed can significantly improve the performance of the nucleic acid hybridisation assay and paves the way for the development of highly sensitive and specific integrated label-free biosensors.

Heat shock proteins are produced when the organisms are exposed to various environmental stress conditions such as temperature, light, toxins. It is a known fact that in bacteria, which has the HSP gene, antibiotics can trigger the expression of the heat shock protein. However, the response of heat shock protein genes to antibiotics has not been fully clarified in the literature yet, studies are still ongoing. In this study, a novel way for the detection heat shock protein65 was investigated using the electrochemical impedance spectroscopy due to its sensitivity, selectivity, low cost. To do so, heat shock protein65 probe and target were designed and the hybridization behavior of the probe with designed target was studied upon the binding onto screen-printed electrodes. Cyclic voltammetry was performed to analyze surface characterization of secreen printed electrodes and the performance of the screen printed electrodes was tested using electrocehimcal impedance spectroscopy by measuring changes in the charge transfer resistance upon hybridization. Based on obtained results, the designed heat shock protein65 probe was confirmed and an appropriate increase in charge transfer resistance values compliance to the literature proved that the electrochemical impedance spectroscopy can be effectively used to detect heast shock protein65 probe label-free. Results presented here can lead to development of antibiotic susceptibility assay based on the heat shock protein genes in future.

Core–shell gold nanorod (AuNR)@silver (Ag) nanostructures with their unique properties have gained enormous interest and are widely utilized in various applications including sensor systems, catalytic reactions, diagnosis, and therapy. Despite the recent progress, simple, effective, low-cost, and easy-to-tune strategies are heavily required to fabricate these nanoparticles (NP) systems. For this, we propose the employment of the polymer of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) as a ligand molecule. A conformal thin layer of polymer of L-DOPA (PLDOPA) with its various functional groups enabled the reduction of silver ions onto the AuNRs and stabilization of the resultant NPs without using any surfactant, reducing agent, and seed material. The shape and growth model of the AuNR@Ag nanostructures was manipulated by simply tuning the amount of silver ions. This procedure created different NP morphologies ranging from concentric to acentric/island shape core–shell nanostructures. Also, even at the highest Ag deposition, the PLDOPA layer is still conformally present onto the Au@Ag core–shell NRs. The unique properties of NP systems provided remarkable characteristics in surface-enhanced Raman spectroscopy, catalytic activity, and cell viability tests.

Successful lysis of cells/microorganisms is a key step in the sample preparation in fields like molecular biology, bioengineering, and biomedical engineering. This study therefore aims to investigate the lysis of bacteria on-chip and its dependence on both microfluidic channel structure and flow rate. Effects of temperature on lysis on-chip were also investigated. To perform these investigations, three different microfluidic chips were designed and produced (straight, zigzag and circular configurations), while the length of the channels were kept constant. As an exemplary case, Mycobacterium smegmatis was chosen to represent the acid-fast bacteria. Bacterial suspensions of 1.5 McFarland were injected into the chips at various flow rates (0.6-8 μl/min) either at room temperature or 50° C. In order to understand the on-chip lysis performance fully, off-chip experiments were carried out at durations which are equal to those bacteria spent in the channel from inlet to the outlet at different flow rates. We also performed COMSOL multiphysics program simulations to evaluate further the effect of the applied parameters. As a result, we found that the structure and the flow rate do not affect lysis over all in all investigated channel types, however on-chip experiments at room temperature produced more effective lysis compared to the on-chip and the off-chip samples performed at higher temperatures. Interestingly on-chip experiments at higher tempratures do not result in effective lysis.

Today, the emergence of antibiotic‐resistance in pathogenic bacteria is considered an important problem for society. Excessive consumption of antibiotics, long‐term treatments, and inappropriate prescriptions continually increase the severity of the problem. Improving antibiotic stewardship requires improved diagnostic testing and therefore, in vitro antibiotic susceptibility testing (AST) is becoming increasingly important. This research details the development of an antibiotic susceptibility test for Mycobacterium smegmatis using Streptomycin as an antibiotic. This strain was selected because it is a member of the slow growing Mycobacterium genus and serves as a useful surrogate organism for Mycobacterium tuberculosis. A commercially available and low‐cost screen‐printed gold electrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome were employed to monitor M. smegmatis nucleic acid sequences using the techniques of Square‐Wave Voltammetry (SWV) and Electrochemical Impedance Spectroscopy (EIS). The results show that it was possible to detect M. smegmatis sequences and distinguish antibiotic‐treated cells from untreated cells with a label‐free molecular detection. As a result, the in vitro antibiotic susceptibility test revealed that M. smegmatis showed sensitivity to streptomycin after a 24 hour incubation, with the developed protocol representing a potential approach to determining antibiotic susceptibility more quickly and economically than current methods. This article is protected by copyright. All rights reserved