Isothermal Amplification of Nucleic Acids Coupled with Nanotechnology and Microfluidic Platforms for Detecting Antimicrobial Drug Resistance and Beyond.

Antibiotic resistance is one of the serious health-threatening issues globally, the control of which is indispensable for rapid diagnosis and treatment because of the high prevalence and risks of pathogenicity. Traditional and molecular techniques are relatively expensive, complex, and non-portable, requiring facilities, trained personnel, and high-tech laboratories. Widespread and timely-detection is vital to the better crisis management of rapidly spreading infective diseases, especially in low-tech regions and resource-limited settings. Hence, the need for inexpensive, fast, simple, mobile, and accessible point-of-care (POC) diagnostics is highly demanding. Among different biosensing methods, the isothermal amplification of nucleic acids is favorite due to their simplicity, high sensitivity/specificity, rapidity, and portability, all because they require a constant temperature to work. Isothermal amplification methods are utilized for detecting various targets, including DNA, RNA, cells, proteins, small molecules, ions, and viruses. In this paper, we discuss various platforms, applications, and potentials of isothermal amplification techniques for biosensing of antimicrobial resistance. We also evaluate the potential of these methods, coupled with the novel and rapidly-evolving platforms offered by nanotechnology and microfluidic devices.

On-Site Detection of Carcinoembryonic Antigen in Human Serum.

Real-time connectivity and employment of sustainable materials empowers point-of-care diagnostics with the capability to send clinically relevant data to health care providers even in low-resource settings. In this study, we developed an advantageous kit for the on-site detection of carcinoembryonic antigen (CEA) in human serum. CEA sensing was performed using cellulose-based lateral flow strips, and colorimetric signals were read, processed, and measured using a smartphone-based system. The corresponding immunoreaction was reported by polydopamine-modified gold nanoparticles in order to boost the signal intensity and improve the surface blocking and signal-to-noise relationship, thereby enhancing detection sensitivity when compared with bare gold nanoparticles (up to 20-fold in terms of visual limit of detection). Such lateral flow strips showed a linear range from 0.05 to 50 ng/mL, with a visual limit of detection of 0.05 ng/mL and an assay time of 15 min. Twenty-six clinical samples were also tested using the proposed kit and compared with the gold standard of immunoassays (enzyme linked immunosorbent assay), demonstrating an excellent correlation (R = 0.99). This approach can potentially be utilized for the monitoring of cancer treatment, particularly at locations far from centralized laboratory facilities.

A novel enhanced dot blot immunoassay using colorimetric biosensor for detection of Toxoplasma gondii infection.

This study reports development of a novel point of care assay, namely an enhanced immuno-dot blot assay, for discrimination of anti-Toxoplasma IgG and anti-Toxoplasma IgM antibodies. This method has been designed based on formation of a sandwich complex between a gold nanoprobe (chitosan gold nanoparticle-anti-human IgG or anti-IgM) and anti- Toxoplasma lysate antigen (TLA) which holds anti-TLA antibodies, either IgG or IgM. Briefly, anti-human IgG or anti-IgM antibody was conjugated to chitosan gold nanoparticles via glutaraldehyde chemistry. Then, lysate antigen was immobilized on the surface of nitrocellulose membrane, which followed by addition of the sera sample and gold nanoprobes. The positive signals were readily detectable via observation with naked eye. This positive color change was further intensified via gold enhancement chemistry. The intensity of biosensor signal was proportional to the concentration of active antibodies on the surface of nanoparticles, titer of T. gondii antibodies in the sera samples and concentration of Toxoplasma lysate antigen coated on the nitrocellulose membrane. A minimum concentration to use the antibodies for conjugation, to detect titer of Toxoplasma IgG and IgM antibodies, and the concentration of TLA coated in nitrocellulose membrane were 0.5 mg/mL, 2 IU/mL, 10 IU/mL, and 20 µg/mL, respectively. This enhanced immuno-dot blot assay offers a simple diagnostic technique without expensive equipment requirement for distinguishing of anti- T. gondii IgM and IgG antibodies in field conditions, pregnant women, and immunocompromised patients.

Solvent effect on the absorption and emission spectra of carbon dots: evaluation of ground and excited state dipole moment.

BACKGROUND: Carbon dots (C-dots) are photoluminescent nanoparticles with less than 10 nm in size. Today, many studies are performed to exploit the photoluminescence (PL) property of carbon dots, and our focus in this study is to estimate the dipole moment of carbon dots. For reaching our aims, C-dots were synthesized and dissolved in the different solvents. RESULTS: Carbon dots with intense photoluminescence properties have been synthesized by a one-step hydrothermal method from a carbon bio-source. In this research, we report on the effect of aprotic solvents on absorption and fluorescence spectra and dipole moments of C-dots dispersed in a range of many aprotic solvents with various polarity and dielectric constant at room temperature. The change in the value of dipole moment was estimated by using the Stokes shifts. The difference between the dipole moment of the excited state and the ground state was shown using an extended form of Lippert equations by Kawski and co-workers. CONCLUSIONS: The values found for µg = 1.077 D, and µe = 3.157 D, as well as the change in the dipole moments. The results showed that the dipole moment of the excited state is more than the ground state, indicating a high density and redistribution of electrons in the excited state. Finally, the quantum yield of C-dots in the eclectic aprotic solvents was communicated and discussed.

(Nano)tag-antibody conjugates in rapid tests.

Antibodies (Abs) are naturally derived materials with favorable affinity, selectivity, and fast binding kinetics to the respective antigens, which enables their application as promising recognition elements in the development of various types of biosensors/bioassays, especially in rapid tests. These tests are low-cost and easy-to-use biosensing devices with broad applications including medical or veterinary diagnostics, environmental monitoring and industrial usages such as safety and quality analysis in food, providing on-site quick monitoring of various analytes, making it possible to save analysis costs and time. To reach such features, the conjugation of Abs with various nanomaterials (NMs) as tags is necessary, which range from conventional gold nanoparticles to other nanoparticles recently introduced, where magnetic, plasmonic, photoluminescent, or multi-modal properties play a critical role in the overall performance of the analytical device. In this context, to preserve the Ab affinity and provide a rapid response with long-term storage capability, the use of efficient bio-conjugation techniques is critical. Thanks to their prominent role in rapid tests, many studies have been devoted to the design and development of Abs-NMs conjugates with various chemistries including passive adsorption, covalent coupling, and affinity interactions. In this review, we present the state-of-the-art techniques allowing various Ab-NM conjugates with a special focus on the efficiency of the developed probes to be employed in in vitro rapid tests. Challenges and future perspectives on the development of Ab-conjugated nanotags in rapid diagnostic tests are highlighted along with a survey of the progress in commercially available Ab-NM conjugates.

Effect of green GO/Au nanocomposite on in-vitro amplification of human DNA.

Recently nanomaterials have attracted interest for increasing efficiency of polymerase chain reaction (PCR) systems. Here, the authors report on the usefulness of green graphene oxide/gold (GO/Au) nanocomposites for enhancement of PCR reactions. In this study, green GO/Au nanocomposite was prepared with Matricaria chamomilla extract as reducing/capping agent for site-directed nucleation of Auo atoms on surface of GO sheets. The as-prepared green GO/Au nanocomposites were then characterised with UV-VIS spectrophotometer and scanning electron microscopy. Later, the effect of these nanocomposites was studied on end-point and real-time PCR employed for amplification of human glyceraldehyde-3-phosphate dehydrogenase gene. The results indicated that GO/Au nanocomposite can improve both end-point and real-time PCR methods at the optimum concentrations, possibly through interaction between GO/Au nanocomposite and the materials in PCR reaction, and through providing increased thermal convection by the GO surface as well as the Au nanostructures. In conclusion, it can be suggested that green GO/Au nanocomposite is a biocompatible and eco-friendly candidate as enhancer of in-vitro molecular amplification strategies.

Surface Plasmon Enhanced Light Scattering Biosensing: Size Dependence on the Gold Nanoparticle Tag.

Surface plasmon enhanced light scattering (SP-LS) is a powerful new sensing SPR modality that yields excellent sensitivity in sandwich immunoassay using spherical gold nanoparticle (AuNP) tags. Towards further improving the performance of SP-LS, we systematically investigated the AuNP size effect. Simulation results indicated an AuNP size-dependent scattered power, and predicted the optimized AuNPs sizes (i.e., 100 and 130 nm) that afford extremely high signal enhancement in SP-LS. The maximum scattered power from a 130 nm AuNP is about 1700-fold higher than that obtained from a 17 nm AuNP. Experimentally, a bio-conjugation protocol was developed by coating the AuNPs with mixture of low and high molecular weight PEG molecules. Optimal IgG antibody bioconjugation conditions were identified using physicochemical characterization and a model dot-blot assay. Aggregation prevented the use of the larger AuNPs in SP-LS experiments. As predicted by simulation, AuNPs with diameters of 50 and 64 nm yielded significantly higher SP-LS signal enhancement in comparison to the smaller particles. Finally, we demonstrated the feasibility of a two-step SP-LS protocol based on a gold enhancement step, aimed at enlarging 36 nm AuNPs tags. This study provides a blue-print for the further development of SP-LS biosensing and its translation in the bioanalytical field.

State-of-the-Art and Trends in Synthesis, Properties, and Application of Quantum Dots-Based Nanomaterials.

Quantum dots (QDs) with a nanoscale size range have attracted significant attention in various areas of nanotechnology due to their unique properties. Different strategies for the synthesis of QD nanoparticles are reported in which various factors, such as size, impurities, shape, and crystallinity, affect the QDs fundamental properties. Consequently, to obtain QDs with appropriate physical properties, it is required to select a synthesis method which allows enough control over the surface chemistry of QDs through fine-tuning of the synthesis parameters. Moreover, QDs nanocrystals are recently used in multidisciplinary research integrated with biological interfaces. The state-of-the-art methods for synthesizing QDs and bioconjugation strategies to provide insight into various applications of these nanomaterials are discussed herein.

Biomimetic synthesis of silver nanoparticles using Matricaria chamomilla extract and their potential anticancer activity against human lung cancer cells.

Herbs having various natural substances can be utilized for the biosynthesis of Silver nanoparticles (AgNPs) and act as a stable, reliable and biocompatible alternative instead of the current physical and chemical approaches. It has been reported that Matricaria chamomilla possesses unique properties, especially anti-cancerous effects. The objective of the current work was to assess the chemical characteristics and anticancer effects of biosynthesized AgNPs applying aqueous extracts of M. chamomilla against A549 lung cancer cells. UV-visible spectrum showed the maximum absorption of the biosynthesized AgNPs at 430 nm. The crystalline structure of biosynthesized AgNPs in optimal conditions was confirmed by XRD. Moreover, the presence of Ag as the ingredient element was exhibited via EDX analysis. FT-IR results also verified the AgNPs synthesis using a plant extract. The spherical shapes of the AgNPs with an average diameter size around 45.12 nm and a zeta potential value of -34 mV were characterized using DLS, and confirmed through FE-SEM and TEM. In vitro cytotoxicity assay using MTT revealed that the biosynthesized AgNPs exhibited a dose- and time- dependent cytotoxic effect against A549 lung cancer cells. Moreover, the apoptotic effects of the AgNPs were demonstrated using DAPI staining, real-time PCR and flow cytometry. According to these findings, using M. chamomilla in combination with AgNPs via green-synthesis approach may be an efficient strategy for effective treatment of lung cancer.

Anti-CD24 bio Modified PEGylated Gold Nanoparticles as Targeted Computed Tomography Contrast Agent.

Purpose: Molecular imaging is one of the import methods for recognition of cancer at the early stage in order to enhance the capacity of remedy. This study was aimed to introduce a new contrast agent that was targeted with CD24 so as to improve the CT scan detection of cancer cells with higher CD24 expression. Methods: The surface modifications of gold nanoparticles (Au-NPs) were done with long PEG (HS-PEG-CH3O) and short PEG (HS-PEG-COOH) chains to enhance their stability and capacity for immobilization of different antibodies. MTT assay was carried out to assess the biocompatibility of the NPs. The obtained contrast agent was implemented in the targeted CT imaging based on in vitro and in vivo studies of breast cancer. Results: The results revealed that the attached CD24 to the cell surface of PEGylated Au-NPs could enhance significantly the cells CT number (40.45 HU in 4T1, while it was 16.61 HU in CT26) It was shown that the attenuation coefficient of the molecularly targeted cells was more than 2 times excessive than the control groups. Further, the tumor region in model of xenograft tumor has higher density compare to the omnipaque groups, 60 min after injection (45 Hu vs.81 Hu). These results showed that the nanoparticles stayed in tumor region for longer time. Conclusion: It is predicted that the synthesized nanoparticle can be used as computed tomography contrast agent. Also, it can be used to identify the tumor cells with higher expression of CD24 at the early stages more efficiently compare to the other routine methods.

Watercress-based electrospun nanofibrous scaffolds enhance proliferation and stemness preservation of human adipose-derived stem cells.

The present study describes the effects of Watercress extract (WE) based electrospun nanofibrous mats on the regulation of adhesion, proliferation, cytoprotection and stemness preservation of adipose-derived stem cells (ADSCs). Watercress (Nasturtium officinale) is one of the most important medicinal plant with a board spectrum of biological functions. For this purpose, WE-loaded PCL-PEG nanofibers were fabricated by electrospinning and characterized using FE-SEM and FTIR. Adhesion, proliferation and cytoprotection of ADSCs on the nanofibers was investigated using FE-SEM and MTT assays. Analysis of cell cycle was carried out by flow-cytometry. Finally, qPCR was applied to measure the expression levels of cell cycle-regulated genes and stemness markers of ADSCs grown on the nanofibers. In this study, we found that WE-loaded PCL-PEG nanofibers had great antioxidant potential and exhibited higher cytoprotection, better adhesion, and significantly increased proliferation of ADSCs. The greater proliferation and preserving stemness ability of ADSCs on WE based nanofibers was further confirmed by higher expression levels of cell cycle-regulated genes and stemness markers. These results demonstrate that WE-loaded PCL-PEG electrospun nanofibrous mats appear suitable to support ADSCs adhesion and proliferation while concurrently preserving the cell stemness, therefore representing a hopeful approach for applying in stem cell based regenerative medicine.

Emerging Importance of Phytochemicals in Regulation of Stem Cells Fate via Signaling Pathways.

To reach ideal therapeutic potential of stem cells for regenerative medicine purposes, it is essential to retain their self-renewal and differentiation capacities. Currently, biological factors are extensively used for stemness maintaining and differentiation induction of stem cells. However, low stability, high cost, complicated production process, and risks associated with viral/endotoxin infection hamper the widespread use of biological factors in the stem cell biology. Moreover, regarding the modulation of several signaling cascades, which lead to a distinct fate, phytochemicals are preferable in the stem cells biology because of their efficiency. Considering the issues related to the application of biological factors and potential advantages of phytochemicals in stem cell engineering, there is a considerable increasing trend in studies associated with the application of novel alternative molecules in the stem cell biology. In support of this statement, we aimed to highlight the various effects of phytochemicals on signaling cascades involved in commitment of stem cells. Hence, in this review, the current trends in the phytochemicals-based modulation of stem cell fate have been addressed. Copyright © 2017 John Wiley & Sons, Ltd.

Ultrasensitive Detection of Cancer Prognostic miRNA Biomarkers Based on Surface Plasmon Enhanced Light Scattering.

The development of simple yet ultrasensitive biosensing approaches for the detection of cancer prognostic microRNA is an important step toward their successful clinical implementation. We demonstrate the relevance for the detection of circulating miRNA of a novel signal amplification scheme based on surface plasmon resonance enhanced light scattering (SP-LS). In addition to experimental optimization carried out using gold nanoparticle (AuNP) tags conjugated with a monoclonal antibody with high affinity for RNA*DNA hybrid duplexes, simulation modeling was conducted to obtain insights about SP-LS biosensing. SP-LS enabled the detection of miRNA-122 at subpicomolar concentrations within 30 min, and a limit of detection of 2 attomoles (60 fM, 50 µL) was determined. MiRNA-122 could also be reliably detected in a high concentration background of nontarget miRNA. The proposed SP-LS miRNA detection approach could be readily applied to other miRNA targets of diagnostic importance and further developed to allow for multiplex measurements of miRNA panels. The promising results obtained in this study and advantageous features of SP-LS warrant further development and its application to clinical samples.

Cloning and Expression of Recombinant Human Endostatin in Periplasm of Escherichia coli Expression System.

PURPOSE: Recombinant human endostatin (rhEs) is an angiogenesis inhibitor which is used as a specific drug in the treatment of non-small-cell lung cancer. In the current research, we developed an efficient method for expressing soluble form of the rhEs protein in the periplasmic space of Escherichia coli via fusing with pelB signal peptide. METHODS: The human endostatin (hEs) gene was amplified using synthetic (hEs) gene as a template; then, cloned and expressed under T7 lac promoter. IPTG was used as an inducer for rhEs expression. Next, the osmotic shock was used to extraction of protein from the periplasmic space. The presence of rhEs in the periplasmic space was approved by SDS-PAGE and Western blotting. RESULTS: The results show the applicability of pelB fusion protein system usage for secreting rhEs in the periplasm of E. coli in the laboratory scale. The rhEs represents approximately 35 % (0.83mg/l) of the total cell protein. CONCLUSION: The present study apparently is the first report of codon-optimized rhEs expression as a fusion with pelB signal peptide. The results presented the successful secretion of soluble rhEs to the periplasmic space.

Design and development of PCR-free highly sensitive electrochemical assay for detection of telomerase activity using Nano-based (liposomal) signal amplification platform.

Telomerase, which has been detected in almost all kinds of cancer tissues, is considered as an important tumor marker for early cancer diagnostics. In the present study, an electrochemical method based on liposomal signal amplification platform is proposed for simple, PCR-free, and highly sensitive detection of human telomerase activity, extracted from A549 cells. In this strategy, telomerase reaction products, which immobilized on streptavidin-coated microplate, hybridized with biotinylated capture probes. Then, dopamine-loaded biotinylated liposomes are attached through streptavidin to biotinylated capture probes. Finally, liposomes are ruptured by methanol and the released-dopamine is subsequently measured using differential pulse voltammetry technique by multi-walled carbon nanotubes modified glassy carbon electrode. Using this strategy, the telomerase activity extracted from 10 cultured cancer cells could be detected. Therefore, this approach affords high sensitivity for telomerase activity detection and it can be regarded as an alternative to telomeric repeat amplification protocol assay, having the advantages of simplicity and less assay time.

Toward Intraoperative Detection of Disseminated Tumor Cells in Lymph Nodes with Silicon Nanowire Field Effect Transistors.

Within an hour, as little as one disseminated tumor cell (DTC) per lymph node can be quantitatively detected using an intraoperative biosensing platform based on silicon nanowire field-effect transistors (SiNW FET). It is also demonstrated that the integrated biosensing platform is able to detect the presence of circulating tumor cells (CTCs) in the blood of colorectal cancer patients. The presence of DTCs in lymph nodes and CTCs in peripheral blood is highly significant as it is strongly associated with poor patient prognosis. The SiNW FET sensing platform out-performed in both sensitivity and rapidity not only the current standard method based on pathological examination of tissue sections but also the emerging clinical gold standard based on molecular assays. The possibility to achieve accurate and highly sensitive analysis of the presence of DTCs in the lymphatics within the surgery time frame has the potential to spare cancer patients from an unnecessary secondary surgery, leading to reduced patient morbidity, improving their psychological wellbeing and reducing time spent in hospital. This study demonstrates the potential of nanoscale field-effect technology in clinical cancer diagnostics.

Gold Nanoprobe-Based Detection of Human Telomerase Reverse Transcriptase (hTERT) Gene Expression.

Human Telomerase Reverse Transcriptase (hTERT) gene is expressed in all types of cancers, and it is considered as unique biomarker for early detection, monitoring and prognosis of different cancers. Routinely, the main techniques for detection of hTERT gene expression are based on enzymatic amplifications which need specified equipments, expert personnel and high cost and time. With regarding to the clinical importance of analysis of hTERT gene expression, we have developed a rapid, simple and low cost method which detects hTERT RNA target in 5 µl reaction scale using gold nanoprobes. The method is based on the inhibition of nanoparticle aggregation in the presence of MgCl 2 and it can be used as a basic technique for development of clinical scale gold nanoprobe nanobiodiagnostics for detection of hTERT gene expression with a limit of detection at fmol/µl concentrations.

Clofarabine Has Apoptotic Effect on T47D Breast Cancer Cell Line via P53R2 Gene Expression.

PURPOSE: Clofarabine, a purine nucleoside analogue and inhibitor of Ribonucleotide Reductase (RR), is used for treatment of leukemia. Clofarabine-induced defect in DNA replication, induces p53 and subsequently P53R2 genes as subunit of RR. clofarabine deregulated P53R2 gene expression leading to the elevated levels of P53R2 which impose resistance to DNA damaging drugs. In this study the apoptotic and cytotoxic effects of clofarabine has been investigated on breast cancer cell line. METHODS: Cofarabine cytotoxicity on T47D cells has been studied by MTT assay. T47D cells were exposed to the different concentrations of clofarabine for 24, 48 and 72 hours intervals. Relative expression of P53R2 gene has been studied using real-time PCR. Moreover, after treating with clofarabine the apoptotic and necrotic cells were detected using Annexin V and propodium iodide (PI) reagents by flowcytometry technique. RESULTS: MTT assay results showed that the clofarabine IC50 on T47D cell line were 3 and 2.5µM after 48 and 72 h exposure, respectively. Clofarabine did not show any significant cytotoxic effect after 24 h exposure. The analysis of qRT-PCR showed a significant increase in P53R2 gene expression in treated cells with both 2.5 and 3 µM doses and also, the results of flowcytometry revealed 26.91 and 74.46 percent apoptosis induction in 48 and 72h treatments respectively in comparison to the control groups. CONCLUSION: Our results showed that apoptotic and cytotoxic effects of clofarabine on T47D cell line were in time and dose dependent manner; therefore it could be considered a new candidate in breast cancer therapy.

Complementary metal oxide semiconductor compatible silicon nanowires-on-a-chip: fabrication and preclinical validation for the detection of a cancer prognostic protein marker in serum.

An integrated translational biosensing technology based on arrays of silicon nanowire field-effect transistors (SiNW FETs) is described and has been preclinically validated for the ultrasensitive detection of the cancer biomarker ALCAM in serum. High-quality SiNW arrays have been rationally designed toward their implementation as molecular biosensors. The FET sensing platform has been fabricated using a complementary metal oxide semiconductor (CMOS)-compatible process. Reliable and reproducible electrical performance has been demonstrated via electrical characterization using a custom-designed portable readout device. Using this platform, the cancer prognostic marker ALCAM could be detected in serum with a detection limit of 15.5 pg/mL. Importantly, the detection could be completed in less than 30 min and span a wide dynamic detection range (∼10(5)). The SiNW-on-a-chip biosensing technology paves the way to the translational clinical application of FET in the detection of cancer protein markers.

Curcumin inhibits leptin gene expression and secretion in breast cancer cells by estrogen receptors.

BACKGROUND: Recent studies suggested that leptin as a mitogenic factor might play an important role in the process of initiation and progression of human cancer. Therefore, it could be considered as a target for breast cancer therapy. A previous study has showed that expression of leptin gene could be modulated by activation of estrogen receptors. Curcumin is a diferuloylmethane that has been shown to interfere with multiple cell signaling pathways and extensive research over the last 50 years has indicated this polyphenol can both prevent and treat cancer. Based on the fact that targeting of leptin could be considered as a novel strategy for breast cancer therapy, the aim of this study is the investigation of potentiality of curcumin for inhibition of leptin gene expression and secretion, and also, its link with expression of estrogen receptors. METHODS: Cytotoxic effect of curcumin on T47D breast cancer cells was investigated by MTT assay test after 24 and 48 treatments. Thereafter, the cells treated with different concentrations of curcumin. The levels of leptin, estrogen receptor α and estrogen receptor ß genes expression was measured in the treated and control cells by Reverse-transcription real-time PCR. Amount of secreted leptin in the culture medium was also determined by ELISA in both treated and untreated cells. Finally data were statistically analyzed by one-way ANOVA test. RESULTS: Analysis of MTT assay data showed that curcumin inhibits growth of T47D cells with dose dependent manner. There were also significant difference between control and treated cells in the levels of leptin, estrogen receptor α expression levels and the quantity of secreted leptin that both were decreased in the treated cells compared with control cells. CONCLUSION: Based on the results, curcumin inhibits the expression and secretion of leptin and it could probably be used as a drug candidate for the breast cancer therapy through the leptin targeting in the future.