An ultrasensitive genosensor for detection of toxigenic and non-toxigenic Clostridioides difficile based on a conserved sequence in surface layer protein coding gene.

Clostridioides difficile (C. difficile) is the most common agent of antibiotic-associated diarrhea, leading to intestinal infection through the secretion of two major toxins. Not all strains of this bacterium are toxigenic, but some of them cause infection via their accessory virulence factors, such as surface layer protein (SlpA). SlpA is conserved in both toxigenic and non-toxigenic strains of C. difficile. In the present work, an amplification-free electrochemical genosensor was designed for the detection of the slpA gene. A glassy carbon electrode coated with gold nanoparticle-reduced graphene oxide nanocomposite was used as the working electrode, and its surface was modified using a simple thiolated linear oligonucleotide as the bioreceptor. Moreover, the hexaferrocenium tri[hexa(isothiocyanato) iron(III)] trihydroxonium (HxFc) complex was used as an intercalator, and its redox signal was recorded using differential pulse voltammetry. Scan rate studies indicated a quasi-reversible adsorption-controlled process for the HxFc complex. This genosensor showed high sensitivity with a limit of detection of 0.2 fM, a linear response range of 0.46-1900 fM, and a satisfactory specificity toward the synthetic slpA target gene. Also, the genosensor indicated responses in the mentioned linear range toward the genome extracted from either toxigenic or non-toxigenic strains of C. difficile.

C. difficile biomarkers, pathogenicity and detection.

BACKGROUND: Clostridioides difficile infection (CDI) is the main etiologic agent of antibiotic-associated diarrhea. CDI contributes to gut inflammation and can lead to disruption of the intestinal epithelial barrier. Recently, the rate of CDI cases has been increased. Thus, early diagnosis of C. difficile is critical for controlling the infection and guiding efficacious therapy. APPROACH: A search strategy was set up using the terms C. difficile biomarkers and diagnosis. The found references were classified into two general categories; conventional and advanced methods. RESULTS: The pathogenicity and biomarkers of C. difficile, and the collection manners for CDI-suspected specimens were briefly explained. Then, the conventional CDI diagnostic methods were subtly compared in terms of duration, level of difficulty, sensitivity, advantages, and disadvantages. Thereafter, an extensive review of the various newly proposed techniques available for CDI detection was conducted including nucleic acid isothermal amplification-based methods, biosensors, and gene/single-molecule microarrays. Also, the detection mechanisms, pros and cons of these methods were highlighted and compared with each other. In addition, approximately complete information on FDA-approved platforms for CDI diagnosis was collected. CONCLUSION: To overcome the deficiencies of conventional methods, the potential of advanced methods for C. difficile diagnosis, their direction, perspective, and challenges ahead were discussed.

Structural and functional alterations of polydopamine-coated hemoglobin: New insights for the development of successful oxygen carriers.

One of the major factors that is currently hindering the development of hemoglobin (Hb)-based oxygen carriers (HBOCs) is the autoxidation of Hb into nonfunctional methemoglobin. Modification with polydopamine (PDA), which is a biocompatible free radical scavenger has shown the ability to protect Hb against oxidation. Due to its tremendous potential in the development of successful HBOCs, herein, we conduct a thorough evaluation of the effect of PDA on the stability, aggregation, structure and function of the underlying Hb. By UV-vis spectrometry we show that PDA can prevent Hb's aggregation while thermal denaturation studies indicate that, although PDA coating resulted in a lower midpoint transition temperature, it was also able to protect the protein from full denaturation. These results are further corroborated by differential scanning calorimetry. Circular dichroism reveals that PDA can promote changes in Hb's secondary structure while, by UV-vis spectroscopy, we show that PDA also interacts with the porphyrin complex located in Hb's hydrophobic pocket. Last but not least, affinity studies show that PDA-coated Hb has a higher capability for oxygen release. Such an effect is further enhanced at lower pH. Importantly, through molecular docking simulations we provide a plausible explanation for the observed experimental results.

Fe3O4/Au/porous Au nanohybrid for efficient delivery of doxorubicin as a model drug.

Fe3O4/Au/porous Au nanohybrids being bi-functional nanoparticles with magnetic properties and high porosity, were synthesized and used for drug delivery. To achieve this purpose, after Fe3O4 nanoparticles synthesis, a gold layer coats them to increase their stability. Then, to improve the loading capacity of Fe3O4/Au nanoparticles, a shell of porous gold was synthesized on the Fe3O4/Au surface by creating an Ag-Au nanohybrid layer on Fe3O4/Au and dissolving the metallic silver atoms in HNO3 (0.01 M). The DLS results show that the synthesized nanohybrid has an average size of 68.0 ± 7.7 nm and a zeta potential of - 28.1 ± 0.2 mV. Finally, doxorubicin (DOX), as a pharmaceutical agent, was loaded onto the Fe3O4/Au/porous Au nanohybrids. The prepared nano-drug enhanced the therapeutic efficacy of DOX on MCF-7 cancer cells compared to the free DOX. These results confirmed a 1.5 times improvement in the antitumor activity of DOX-loaded Fe3O4/Au/porous Au nanohybrids.

MIL-88B(Fe)-reduced graphene oxide as an artificial enzyme for gold nanorod etching and its application to develop the prostate-specific antigen immunosensor.

An immunosensor based on gold nanorods (AuNRs) etchant activity of a metal-organic framework (MOF): MIL-88B(Fe)-reduced graphene oxide (rGMOF) was developed for the determination of prostate-specific antigen (PSA). Several techniques, including FTIR, UV-Vis spectrophotometry, XRD, and electron microscopy, were employed to characterize the MOFs containing iron-oxygen clusters on the surface of reduced graphene oxide. Enzyme mimetic activity of rGMOF before and after bioconjugation with antibodies was calculated as 8.4 and 2.5 U mg-1, respectively. The primary anti-PSA was conjugated to a magnetic bead and used as PSA-specific capturing. Then, the secondary anti-PSA was grafted to the rGMOF. In the presence of antigen, an immuno-sandwich was formed between the conjugations mentioned above. Afterward, AuNRs were etched by rGMOF, and the related spectrum was recorded in the wavelength range 350 to 900 nm. By progressing the etching procedure, the longitudinal LSPR peak of AuNRs was gradually blue-shifted with a linear correlation with the PSA concentration from 0.1 pg mL-1 to 100 ng mL-1. The detection limit was 0.09 pg mL-1. The proposed immunosensor was successfully employed to determine PSA levels in real samples. Since the obtained results showed an excellent correlation with those acquired by the chemiluminescence gold standard method, it has the potential for PSA determination in clinical assays.

Design and Optimization of A Magneto-Plasmonic Sandwich Biosensor for Integration within Microfluidic Devices.

We designed a magneto-plasmonic biosensor for the immunodetection of antigens in minute sample volume. Both spherical gold nanoparticles (AuNP) and magnetic beads (MB) were conjugated to goat anti-rabbit IgG antibody (Ab) capable of recognizing a model target, rabbit IgG (rIgG). The AuNP bioconjugate was used as the optical detection probe while the MB one was used as the capture probe. Addition of the target analyte followed by detection probe resulted in the formation of a sandwich immunocomplex which was separated from the unbound AuNP-Ab conjugate by application of an external magnetic field. The readout was executed either in a direct or in indirect way by measuring the UV-Visible spectrum of each fraction in a specially designed microcell. Dose-response curves were established from the optical signal of the immunocomplex and unbound AuNP-Ab conjugate fractions. Finally, the assay was transposed to a microfluidic cell specially designed to enable easy separation of the immunocomplex and AuNP-Ab conjugate fractions and subsequent analysis of the latter fraction and achieve the quantification of the analyte in the ng/mL concentration range.

High-performance porphyrin-like graphene quantum dots for immuno-sensing of Salmonella typhi.

The extraordinary optical properties of porphyrins have inspired their applications in various fields. Herein, we introduce iron porphyrin bio-mimicked graphene quantum dots (Fe-N-GQDs) as a novel paramagnetic and fluorescent label. The Fe-N-GQD was prepared by the mechanochemical mixing of Fe, N, and C sources followed by pyrolysis at high-temperature and next, the solvothermal treatment was performed. The Fe-N sites in graphene matrix, the structural alterations during the solvothermal treatment, the optical properties, and paramagnetic behaviour were studied using FTIR, Raman and X-ray spectroscopies, and Vibrating sample magnetometer. The structural studies revealed that under solvothermal condition, Fe-N doped graphene sheets cut into ultra-small Fe-N-GQDs containing well-dispersed particles with an average diameter of about 2.5 nm. As a result of Fe-N doping, the photoluminescence quantum yield was enhanced to 86% and strong paramagnetic behaviour was observed. Due to the rich oxygen-containing groups at Fe-N-GQDs surface, it has proper sites for bio-conjugation. The bioconjugated Fe-N-GQDs serve as donors in a prominent fluorescence resonance energy transfer system, while graphene oxide acts as an acceptor. The proposed immunosensor was successfully applied for the detection of Salmonella Typhi Vi antigen in real human serum in the concentration range from 1 pg/mL to 1 µg/mL with the detection limit of 1 pg/mL.

Microfluidic-based synthesized carboxymethyl chitosan nanoparticles containing metformin for diabetes therapy: In vitro and in vivo assessments.

This work was aimed to synthesize novel crosslinked carboxymethyl chitosan nanoparticles (CMCS NPs) containing metformin hydrochloride (MET) using microfluidics (MF) and evaluate their performance for diabetes therapy. The field emission-scanning electron microscopy (FE-SEM) images and dynamic light scattering (DLS) results showed that the NPs average size was 77 ± 19 nm with a narrow size distribution. They exhibited a high encapsulation efficiency (∼90 %) and the controlled drug release while crosslinking using CaCl2. Eventually, the in vivo assessments dedicated an increased body weight up to 7.94 % and a decreased blood glucose level amount of 43.58 % for MF MET-loaded CMCS NPs with respect to the free drug in diabetic rats. Also, the results of histopathological studies revealed the size of the pancreatic islets to be 2.32 µm2 and ß cells intensity to be 64 cells per islet for the diabetic rats after treating with the MF-based sample. These data were close to those obtained for the healthy rats.

Ultrasensitive electrochemical biosensor for detection of microRNA-155 as a breast cancer risk factor.

Due to the stability of microRNAs (miRNAs) in serum and other body fluids, they are known as promising cancer biomarkers. Recent studies have indicated higher expression of miRNA-155 (miR-155) in patients with breast cancer compared to healthy people. In the present report, a rapid and sensitive electrochemical biosensor has been developed for detection of miR-155 as a breast cancer risk factor. At first, a thiolated probe was immobilized on the gold electrode surface. Then, the target (miR-155) was exposed to the probe. In the next step, the positively charged polyethyleneimine-silver nanoparticles as electroactive labels were absorbed onto the negatively charged probe-target hybrid. In the third step, the anodic peak current which was produced due to the oxidation of silver nanoparticles was recorded as the electrochemical signal. The designed biosensor provided an ultrasensitive method for the detection of miR-155 with the detection limit of 20 zmol and a wide linear range from 2 × 10-20 to 2 × 10-12 mol. Moreover, the biosensor was able to detect miR-155 in real serum samples with satisfactory results.

Designing a magnetic inductive micro-electrode for virus monitoring: modelling and feasibility for hepatitis B virus.

A simple model is designed for an inductive immunosensor in which the magnetic particles are attached to the bioreceptors to form a sandwich on the surface of an inductor. The inductor consists of a coil covered on a silicon oxide wafer. The coil comprises 250 turns of a planar gold wire, which is approximately 200 nm thick and 392 mm long, placed in a circle with a diameter of 2 mm. The model is well characterised by controlling the geometrical and electrical parameters and also the permeability of the magnetic material. To evaluate the feasibility of the model for virus monitoring, a novel inductive immunosensor is designed and for the first time applied for the detection of hepatitis B surface antigen (HBsAg). At first, Fab' segment of primary anti-HBsAg is immobilised on the coil. Then, the coil is exposed to HBsAg and the complex is introduced to a secondary antibody conjugated with magnetic particles to form an immune-sandwich. Finally, the influence of magnetic particles on the coil inductance is recorded and used as a signal for HBsAg detection. The magnetic inductive immunosensor showed specific responses toward HBsAg with the detection limit of 1 ng mL-1, linear range of 1 to 200 ng mL-1, and a sensitivity of 6 × 10-4 mL ng-1. The experimental results showed a very good agreement with simulation data indicating the compatibility of sensor sensitivity to the expected theoretical values. Graphical abstract.

Ultrasensitive nano-aptasensor for monitoring retinol binding protein 4 as a biomarker for diabetes prognosis at early stages.

Prognosis of diabetes risk at early stages has become an important challenge due to the prevalence of this disease. Retinol binding protein 4 (RBP4), a recently identified adipokine, has been introduced as a predictor for the onset of diabetes type 2 in coming future. In the present report a sensitive aptasensor for detection of RBP4 is introduced. The immune sandwich was prepared by immobilizing biotinylated RBP4 aptamers on streptavidin coated polystyrene micro-wells and then incubation of RBP4 as target and finally addition of luminol-antibody bearing intercross-linked gold nanoparticles as reporter. The chemiluminescence intensity was recorded in the presence of hydrogen peroxide as oxidant agent and Au3+ as an efficient catalyst for luminol oxidation. The aptasensor responded to RBP4 in the linear concentration range from 0.001 to 2 ng/mL and detection limit was slightly less than 1 pg/mL. The proposed method has successfully applied to determine the RBP4 in patient real serums. By using the intercross-linked gold nanoparticles, it is possible to provide more accessible surface for immobilizing luminol and enhance the chemiluminescence signal. Therefore, the analytical parameters such as sensitivity, specificity, detection limit and linear range were improved in compare to the biosensors reported in the literature.

Simple and rapid method for synthesis of porous gold nanoparticles and its application in improving DNA loading capacity.

Porous gold nanoparticles (GNPs) attracted considerable attention due to their remarkable properties. The porous GNPs due to the high surface area-to-volume ratio have potential applications in areas such as drug delivery, biosensors, and fuel cells. The most frequently used method for synthesis of porous GNPs is the de-alloying approach. Despite the benefits of this approach, the nanoparticles synthesized by this method were not very stable. Nevertheless, we report herein a novel, facile and simple method for synthesis of stable porous GNPs based on Tween 20-capped GNPs (Tween GNPs) and nitric acid. On the other hand, when DNA is loaded to GNPs surface, the resulted conjugates have the potential to be used in different fields such as biomedicine, materials science and especially in nano-biotechnology. Generally, the DNA loading on GNPs is performed using a salt-aging method and its incubating time takes about 24 h. Here, GNPs was replaced by porous GNPs and the incubation time for loading was reduced to 2 h without the needs for tedious salt addition process.

Silver-gold-apoferritin nanozyme for suppressing oxidative stress during cryopreservation.

Reactive oxygen species (ROS) cause oxidative stress, which involves in the pathogenesis of many serious diseases. Apoferittin containing gold-silver nanoparticles (Au-Ag-AFT) was designed and evaluated as a nanozyme for scavenging the ROS. The nanozyme consisting of silver-gold nanohybrid in apoferittin cage represents superoxide dismutase, catalase and peroxidase mimetic activities. The Au-Ag-AFT nanozyme was characterized by spectroscopy, FESEM, TEM and dynamic light scattering. The inhibition process for pyrogallol autoxidation was used for assaying the superoxide dismutase mimetic activity and measuring the kinetic parameters of Au-Ag-AFT nanozyme. Additionally, Aebi method and standard protocol was used for evaluating the catalase and peroxidase mimetic activity. The kcat values for superoxide dismutase, catalase and peroxidase mimetics activity were 1.4 × 106, 0.1 and 9 × 103 s-1 respectively. These values indicated that Au-Ag-AFT nanozyme could act as a suitable ROS scavenger. Additionally, Au-Ag-AFT nanozyme was examined as a protective agent for human sperm against oxidative stress induced during the cryopreservation process. Presence of the nanozyme in the sperm media significantly increased the motility and viability of the cells and also decreased the ROS, apoptosis and necrosis (P < 0.05) compare to the control group.

Long segment detection of HTLV-1 genome based on the fluorescence quenching technique.

Detecting fluorescence changes due to energy transfer between a quencher and fluorophore is a common method used for the fluorescence-based biosensors. In the present report, a new biosensor for long segment detection of the human T cell-lymphotropic virus 1 genome was constructed based on the fluorescence quenching of graphene oxide by gold nanoparticles. The fluorescence signal of unmodified graphene oxide was measured before and after hybridization of target and probes functionalized with gold nanoparticles. The limit of detection of the biosensor was determined to be around 10 pg/mL. The specific design for long segment of target assures the selectivity of biosensor. Our results proposed that further development may be useful to detect other viruses.

A silver(I) doped bud-like DNA nanostructure as a dual-functional nanolabel for voltammetric discrimination of methylated from unmethylated genes.

A small DNA structure, referred to as DNA nanobud (NB), was used for the first time to design a dual-functional nanolabel in order to recognize a particular oligonucleotide sequence, generate and amplify the electrochemical analytical signal. NBs containing numerous repetitive desired sequences were prepared through self-assembly of 8-h rolling circle amplification. Then, redox-active silver ions were loaded onto the NBs by over-night incubation with a solution of AgNO3. The incorporation of Ag+ into NBs was confirmed by field emission scanning electron microscopy, dynamic light scattering, UV-Vis spectroscopy, zeta potential measurements, and energy-dispersive X-ray spectroscopy. A DNA sandwich complex was created after hybridization of Ag+-NB with target sequence, which was captured by immobilized probe on a gold electrode. Cyclic voltammetry was applied to measure the redox signal of silver ions produced typically at a potential around 0.02 V vs. Ag/AgCl. The label can specifically discriminate fully methylated BMP3 gene from fully unmethylated bisulfate-converted part of the gene. The electrochemical signal produced by DNA sandwich complex of gold/probe/BMP3/Ag+-NB was linear toward BMP3 concentration from 100 pM to 100 nM. The method has a 100 pM BMP3 detection limit. Conceivably, this nanolabel can be designed and modified such that it may also be used to detect other sequences with lower detection limits. Graphical abstract Ag+-NB as a new nanolabel for genosensing was formed by loading Ag+ on a spherical DNA nanostructure, nanobud (NB), synthesized by rolling circle amplification process. By using a gold electrode (AuE), Ag+-NB with numerous electroactive cations and binding sites can detect targets and generate amplified electrochemical signals.

A genosensor for detection of HTLV-I based on photoluminescence quenching of fluorescent carbon dots in presence of iron magnetic nanoparticle-capped Au.

Carbon dots and Fe3O4@Au were synthesized to develop a new biosensor to detect DNA target. We investigated the photoluminescence property of carbon dots (CDs) in the presence of Fe3O4-capped Au (Fe3O4@Au). Firstly, we designed two dedicated probes for unique long sequence region of human T-lymphotropic virus type 1 genome. One of the probes was covalently bound to the CDs. In the absence of target, CDs-probe was adsorbed on the surface of Fe3O4@Au through two possible mechanisms, leading to quenching the fluorescence emission of CDs. The fluorescence emission of CDs was recovered in the presence of target since double-stranded DNA cannot adsorb on the Fe3O4@Au. Also, Fe3O4@Au can adsorb the unhybridized oligonucleotides and improves the accuracy of detection. The specificity of the proposed biosensor was confirmed by BLAST search and assessed by exposing the biosensor to other virus targets. The experimental detection limit of the biosensor was below 10 nM with linear range from 10 to 320 nM.

Albumin coated copper-cysteine nanozyme for reducing oxidative stress induced during sperm cryopreservation.

In the present work, SOD mimetic nanozyme (NACu-Cys) consisting of Cu-Cys complex and nano-albumin (NA) were synthesized. After characterizing the nanozyme, its superoxide dismutase (SOD) behavior was evaluated by inhibition of the pyrogallol autoxidation method. The results revealed that NACu-Cys exhibited SOD mimetic activity with a half inhibition concentration (IC50) value of 7.0 × 10-3 µM and a turnover number (kcat) of 5.4 × 107 s-1. In the next step, this nanozyme was applied as a protective agent against oxidative stress induced by sperm cryopreservation. Increasing the motility, raising the viability and reducing the apoptosis occurred as a result of NACu-Cys additions to human sperm freezing medium. Comparison between the natural SOD and SOD mimic behavior of NACu-Cys revealed that this nanoparticle has the ability to be used as oxidative stress decrescent during cryopreservation process.

Ultrasensitive interdigitated capacitance immunosensor using gold nanoparticles.

Immunosensors based on interdigitated electrodes (IDEs), have recently demonstrated significant improvements in the sensitivity of capacitance detection. Herein, a novel type of highly sensitive, compact and portable immunosensor based on a gold interdigital capacitor has been designed and developed for the rapid detection of hepatitis B surface antigen (HBsAg). To improve the efficiency of antibody immobilization and time-saving, a self-assembled monolayer (SAM) of 2-mercaptoethylamine film was coated on IDEs. Afterwards, carboxyl groups on primary antibodies were activated through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and were immobilized on amino-terminated SAM for better control of the oriented immobilization of antibodies on gold IDEs. In addition, gold nanoparticles conjugated with a secondary antibody were used to enhance the sensitivity. Under optimal conditions, the immunosensor exhibited the sensitivity of 0.22 nF.pg ml-1, the linear range from 5 pg ml-1 to 1 ng ml-1 and the detection limit of 1.34 pg ml-1, at a signal-to-noise ratio of 3.

Ultrasensitive optical biosensor for detection of miRNA-155 using positively charged Au nanoparticles.

An ultrasensitive optical biosensor for microRNA-155 (miR-155) was developed to diagnose breast cancer at early stages. At first, the probe DNA covalently bind to the negatively charged gold nanoparticles (citrate-capped AuNPs). Then, the target miR-155 electrostatically adsorb onto the positively charged gold nanoparticles (polyethylenimine-capped AuNP) surface. Finally, by mixing citrate-capped AuNP/probe and polyethylenimine-capped AuNP/miR-155, hybridization occurs and the optical signal of the mixture give a measure to quantify the miR-155 content. The proposed biosensor is able to specify 3-base-pair mismatches and genomic DNA from target miR-155. The novelty of this biosensor is in its ability to trap the label-free target by its branched positively charged polyethylenimine. This method increases loading the target on the polyethylenimine-capped AuNPs' surface. So, proposed sensor enables miR-155 detection at very low concentrations with the detection limit of 100 aM and a wide linear range from 100 aM to 100 fM.

Albumin coated cadmium nanoparticles as chemotherapeutic agent against MDA-MB 231 human breast cancer cell line.

With the aim of dedicating toxicity of cadmium nanoparticles (CdNPs) against invasive breast cancer, with minimum damage to surrounding healthy cells, CdNPs were coated with albumin nanocarrier by nanoprecipitation method and named CdNPs@BSA. The characterization was done by TEM image, DLS and UV-Vis, fluorescence, circular dichroism spectroscopy. The cytotoxic efficacy of the CdNPs@BSA against human breast cancer cells (MDA-MB 231 cells) was examined by MTT assay. Apoptosis, as the mechanism of cell death, was verified by inverted microscopy, fluorescent microscopy, gel electrophoresis and flow cytometry. The role of ROS generation in apoptosis was also studied. It was found that the resulted CdNPs@BSA (diameter of 88 nm and zeta potential of about -18.85 mV) was suitable for penetration in tumour micro vessels. In the form of CdNPs@BSA, the 77% of the secondary structure and almost all of the tertiary structure remain intact. Comparing to CdNPs, CdNPs@BSA could significantly suppress the MDA-MB 231 while they were less toxic on WBCs. Therefore, they could be a brilliant candidate to be used as a chemotherapeutic agent against invasive breast cancer cells.