Nonspecific Sites Research Articles

A highly sensitive label-free amperometric biosensor for norfloxacin detection based on chitosan-yttria nanocomposite

Here, non-invasive and label-free detection of trace-level of norfloxacin (NF) in human urine samples has been reported using the electrochemical technique. Nanostructured yttrium oxide (nY2O3) was synthesized at low-temperature using a one-step hydrothermal process. These nY2O3 were characterized by various methods including XRD, FT-IR, Raman spectroscopy, and TEM. A biosensing platform based on nY2O3 modified with chitosan (CH) was fabricated for the detection of NF. The nanocomposite film (CH-Y2O3/ITO) was characterized by FE-SEM, contact angle measurements, and electrochemical techniques. Further, fluoroquinolones antibodies (anti-FQ) were used to modify the CH-Y2O3/ITO electrode via covalent interaction. Non-specific sites were blocked by bovine serum albumin (BSA), those present on the anti-FQ/CH-Y2O3/ITO electrode surface. The response study of BSA/anti-FQ/CH-Y2O3/ITO bioelectrode towards NF detection revealed a wide range (1 pM-10 μM) with a lower detection limit of 3.87 pM using differential pulse voltammetry (DPV). The sensitivity obtained is as high as 10.14 μA μM−1 cm2 with a fast response time of ~10 min. Moreover, the diagnostic performance of the fabricated sensor was evaluated to detect NF in urine spiked sample. The recovery of NF from the spiked sample was observed from 90.5 to 101.1%, with a maximum relative standard deviation of 7.04. The obtained results of the fabricated bioelectrode (BSA/anti-FQ/CH-Y2O3/ITO) was validated with ELISA. The results were found better when compared with earlier described biosensors and commercially existing ELISA in terms of sensitivity and lower detection limit.

Dynamic Covalent Synthesis of Crystalline Porous Graphitic Frameworks

Summary Porous graphitic framework (PGF) is a two-dimensional (2D) material that has emerging energy applications. An archetype contains stacked 2D layers, the structure of which features a fully annulated aromatic skeleton with embedded heteroatoms and periodic pores. Due to the lack of a rational approach in establishing in-plane order under mild synthetic conditions, the structural integrity of PGF has remained elusive and ultimately limited its material performance. Here, we report the discovery of the unusual dynamic character of the C=N bonds in the aromatic pyrazine ring system under basic aqueous conditions, which enables the successful synthesis of a crystalline porous nitrogenous graphitic framework with remarkable in-plane order, as evidenced by powder X-ray diffraction studies and direct visualization using high-resolution transmission electron microscopy. The crystalline framework displays superior performance as a cathode material for lithium-ion batteries, outperforming the amorphous counterparts in terms of capacity and cycle stability.

Nanostructured transition metal chalcogenide embedded on reduced graphene oxide based highly efficient biosensor for cardiovascular disease detection

Herein, we report nanostructured metal chalcogenide (molybdenum tetraselenide, nMo3Se4) embedded on reduced graphene oxide (rGO) based electrochemical immunosensor for cardiovascular disease biomarker [cardiac troponin I (cTnI)] detection. The nanostructured molybdenum tetraselenide-reduced graphene oxide (nMo3Se4-rGO) was synthesized via low temperature hydrothermal method and further functionalization was carried out using 3-aminopropyltriethoxy silane (APTES). Deposition of functionalized nanostructured material (APTES/nMo3Se4-rGO) was conducted onto hydrolysed indium tin oxide (ITO) coated glass platform by using electrophoretic deposition (EPD) technique at a DC potential of 15 V for 60 s. The APTES/nMo3Se4-rGO/ITO platform was then immobilized with monoclonal anti-cardiac troponin I (anti-cTnI) via EDC-NHS chemistry and bovine serum albumin (BSA) was employed for blocking of non-specific sites. The morphological and functional characterization of synthesized materials and fabricated electrodes were characterized by X-ray diffraction, Scanning electron microscopy, Transmission electron microscopy, Fourier transmission infrared spectroscopy and Atomic force microscopy. The electrochemical characterization and biosensing performance of the fabricated bioelectrodes was investigated by cyclic voltammetry studies. The fabricated biosensing (BSA/anti-cTnI/APTES/nMo3Se4-rGO/ITO) platform shows wider linear detection range [1 fg mL−1–100 ng mL−1] with higher sensitivity [36.2 μA log (mL ng−1) cm−2] and remarkable lower detection limit [1 fg mL−1]. We believe that our fabricated biosensor has potential for the detection of cTnI biomarker and towards the better understanding of cardiovascular disease.

Bacterial Biofilm and Biofilm associated environmental protection

Biofilm is the syntrophic consortium of the microorganisms that mainly comprises of sessile groups of bacterial cells that adhere to the inanimate or living surfaces with the help of extracellular polymeric substance (EPS)and pili by forming a matrix composed of exopolysaccharide, proteins and DNA(polymeric conglomeration). The sessile group of cells forming the biofilm develop intimate connection by Quorum Sensing (QS). EPS forming the biofilm comprises glycocalyx and chemically made up of carbohydrates, proteins and nucleic acid. EPS helps those adherent cells by embedding them within a slimy layer and provides nutrients to the developing cells within the biofilm. Thus, it helps in pathogenesis of biofilm associated with infection and resistance due to the formation of impermeable layers to the penetration of antibiotics and drugs. Biofilm can be formed by the microbes depending on various different factors including cellular recognition of specific or non-specific attachment sites on surface, nutritional cures or by exposure of planktonic cells to sub-inhibitory concentration of antibiotics. Besides this pathogenicity the biofilms are able in bioremediation. In this review we are focused to exploit different strategies which are being used in bioremediation by the biofilms.

EMPLOYMENT OF INDIRECT DOT-ELISA FOR THE DETECTION OF ANTI-PARAMPHISTOMUMEPICLITUM ANTIBODIES IN RUMINANTS

Paramphistomosis is one of the major parasitic diseases causing heavy economic losses to livestock production. Diagnosis of disease in early stage is very important for minimizing the losses through effective treatment. The conventional methods of diagnosis such as detection of eggs in faeces by sedimentation and floatation techniques have limitations while modern serology based tests like enzyme linked immune-sorbent assay requires well equipped laboratory. The immature and adult Paramphistomumepiclitum were collected from rumen of slaughtered goat and buffaloes in slaughterhouses fromBareilly, Delhi, Dehradun and Ludhiana. Collected parasites were thoroughly washed in normal saline and processed separately for antigen preparation. Parasites were homogenized in 0.1M PBS pH=7.4, sonicated in Soniprep-150 for 8 min (4 cycles of 2 min each) and centrifuged at 15000 rpm for 15 min at 4C. The supernatant obtained was filtered through 0.22m Millex–GV filter (Millipore, France) and stored in small aliquots of 0.5 ml each at -20C to be used as somatic antigen for ELISA test. Sera were collected at monthly interval from rabbits immunized by somatic P. epiclitum antigen for determining the titre variation with time and for Dot-ELISAtests. Indirect Dot-ELISA was standardized using antigen concentration ranging from 5μg/μl – 10ng/μl and goat anti-rabbit HRPO conjugate dilutions. P. epiclitum antigen was used for coating nitro cellulose membrane (NCM) pad on combs for DotELISA and kept overnight at 4˚C. The combs were then incubated in 3% lactogen in PBS, pH=7.4, at 37˚C for 1 hr for blocking the non-specific antigen binding sites. Subsequently, the combs were incubated in rabbit anti-P. epiclitum sera in dilution range 1:50 – 1: 90000 at 37˚C for 1 hr followed by three washings in PBS of 2 min each. The combs were then incubated in goat anti-rabbit HRPO conjugate at 37˚C for 1 hr again followed by 4 washings in PBS of 2 min each. The combs were then incubated in substrate 3, 3'- Diamino-benzidine hydrochloride (5 mg/10 ml PBS + 10μl 0.06% H O ) for 2 2 5- 15 min. Development of dark brown spot indicated positive reaction after using various control. The optimum antigen concentration was found to be 100ng/μl and optimum conjugate dilution was found to be 1:500. Anti-sera collected from rabbits at the interval of 30 and 60 days showed a maximum titre of 1:40,000. The experimentally infected sheep sera were taken at weekly interval from four sheep, which were examined against adult somatic P. epiclitum antigen following concentrations 50-75 ng/µl and conjugate dilution 1:500 to 1:1000 and sera dilution at 1:600 and 1:800. Sheep sera showed reaction after 2 weeks post infection. A total of 200 sera samples were examined by indirect Dot-ELISA. Overall percent positive incidence rate was recorded to be 33 percent. The highest percent positivity (38.66%) was found in buffaloes followed by 31.7% in sheep, 26.66% in cattle and 25% in goats. In the present study, a rapid and simple test (Dot-ELISA) was developed for the diagnosis of paramphistomosis. The findings are helpful for detection of paramphistomum antibodies in naturally infected animals and can be used under field conditions.

A highly sensitive fluorescent immunosensor for sensitive detection of nuclear matrix protein 22 as biomarker for early stage diagnosis of bladder cancer.

A novel strategy is reported for highly sensitive, rapid, and selective detection of nuclear matrix protein NMP22 using two-color quantum dots based on fluorescence resonance energy transfer (FRET). Quantum dots (QDs) are highly advantageous for biological imaging and analysis, particularly when combined with (FRET) properties of semiconductor quantum dot (QDs) are ideal for biological analysis to improve sensitivity and accuracy. In this FRET system narrowly dispersed green emitting quantum dot CdTe core is used as a donor and labelled by monoclonal (mAb) antibody, while orange emitting quantum dot CdTe/CdS core shell is used as an accepter and labelled by polyclonal (pAb) antibody. The quantum dots are labelled by antibodies using EDC/NHS as crosslinking agent. Bovine serum albumin (BSA) solution was added to block nonspecific binding sites. The fluorescence intensity of QDs accepter decreased linearly with the increasing concentrations of NMP22 from 2–22 pg mL−1 due to FRET system and fluoroimmunoassay reaction. This method has good regression coefficient (R2 = 0.998) and detection limit was 0.05 pg mL−1. The proposed FRET-based immunosensor provides a quick, simple and sensitive immunoassay tool for protein detection, and can be considered as a promising approach for clinical applications. The proposed FRET-based immunosensor provides a quick, simple and sensitive immunoassay tool for protein detection, and can be considered as a promising approach for clinical applications.

Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.

SummaryElectron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.

Stable thin films of human P53 antigen on gold surface for the detection of tumour associated anti-P53 autoantibodies

An electrochemical impedimetric immunosensor was developed for the label-free detection of autoantibodies against the human P53 antigen (protein) involved in carcinogenesis. The immunosensor was based on the covalent immobilization of human P53-protein (P53ag) using amide coupling onto gold electrode pre-grafted with phenylethylamine-succinic acid (Au-PEA-SA). The non-specific binding sites on the activated Au-PEA-SA were blocked with bovine-serum albumin (BSA) to yield an Au-PEA-SA-P53ag/BSA immunosensor. The gold electrode modification steps were monitored using X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The analytical performance of Au-PEA-SA-P53ag/BSA towards the detection of anti-P53 autoantibodies showed a concentration-dependent impedimetric (RCT) response. A wide linear range (1.0 –1000 ng.mL-1) with a low detection limit of 103 pg.mL−1 and high RCT sensitivity of 0.749 kΩ.ng.mL−1.cm−2 was obtained. The Au-PEA-SA-P53ag/BSA could be regenerated (using dimethylsulfoxide/formamide) several times without the loss of covalently bound human P53ag as a sensing element. The detection signal was retained and about 92% of the original signal was recovered without damage to the immunoelectrode after the 6 regeneration cycles. The real sample detection was investigated using serum recovery method. An excellent % recovery was obtained between 84.8 and 93.7%.

Amino-Acid-Based Ionic Liquids for the Improvement in Stability and Activity of Cytochrome c: A Combined Experimental and Molecular Dynamics Study.

The demand for long-term storage and stability of proteins has increased substantially in the pharmaceutical industries, yet the sensitivity of proteins toward the environment has become a cardinal task for researchers. To deal with this, we have selected a multifunctional enzyme Cytochrome-c (Cyt-c) involved in many chemical and biochemical reactions as model protein, which is very sensitive and loses structural integrity on exposure to the environment. The remarkable features of ionic liquids (ILs) have entitled them as alternatives to aqueous and organic solvents for solubility, storage, and surrogate reaction medium. Hence, we have adapted the biocompatible and nontoxic cation and anion based amino acid ILs (CAAAILs) as potential solvents for storage and stability of Cyt-c. Herein, we report the molecular insights and thermodynamics of interaction between CAAAILs and Cyt-c with the help of isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), UV-vis, CD, and fluorescence spectroscopy as well as molecular docking and molecular dynamics (MD) simulations. The structure and stability of Cyt-c remain unchanged in the presence of CAAAILs. Both electrostatic and hydrophobic interactions are accountable for the binding of CAAAILs in the region between terminal helices and the loop of Cyt-c through nonspecific multiple binding sites, which can be exploited for storage and stability of proteins and will be helpful in designing new biobased ILs for biochemical applications.

Establishment of electrical immunosensor for the detection of nuclear matrix protein-22

Objective A label-free electrochemical immunosensor was developed for the detection of nuclear matrix protein-22 (NMP22) as a biomarker of bladder cancer. Methods The study was based on the establishment and validation of the methodology. Urine samples were collected from 20 patients with bladder cancer and 20 controls in the affiliated Hongqi hospital of Mudanjiang medical university from September in 2017 to July in 2019 to validate the developed method. A screen-printed electrode (SPE) was modified with a film of a composite made from the reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) immobilized Zn-based-Metal-organic frameworks deposited with Au nanoparticles (rGO-TEPA@Au-ZIF8). Primary antibody against NMP22 was immobilized on the Au nanoparticles on the surface of the modified SPE, which then was blocked with bovine serum albumin to elimiate nonspecific binding sites. The process of the construction of the proposed sensorwas characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Differential pulse voltammetry was used to evaluate the linear range, recovery, precision, selectivity and stability. The data were analyzed by Mann-Whitney U test. Results Under optimal conditions, the immunosensor exhibited a linear range of 0.01-1000 ng/mlwith a detection limit of 3.33 pg/ml (S/N=3) and a standard recovery of 97.65%-107.05%. The levels of NMP22 in urine samples from patients with bladder cancer [66.03 (4.34, 91.74)]ng/ml determined by the proposed sensor were significantly higher than those of controls 0.54(0.06, 8.84) ng/ml(P=0.001). Conclusion The immunosensor can achieve sensitive, rapid and acucurate detection of NMP22, and has potential application prospects in monitoring tumor markers. Key words: Urinary bladder neoplasms; Nuclear proteins; Biosensing techniques; Electrochemical techniques; Immunoassay; Graphite; Polyamines; Biomarkers, tumor

A new mixed inhibitor of adenosine deaminase produced by endophytic Cochliobolus sp. from medicinal plant seeds.

Medicinal plants have been studied for potential endophytic interactions and numerous studies have provided evidence that seeds harbor diverse microbial communities, not only on their surfaces but also within the embryo. Adenosine deaminase (ADA) is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Therefore, in this study, 20 types of medicinal plant seeds were used to screen endophytic fungi with tissue homogenate and streak. In addition, 128 morphologically distinct endophyte strains were isolated and their ADA inhibitory activity determined by a spectrophotometric assay. The strain with the highest inhibitory activity was identified as Cochliobolus sp. Seven compounds were isolated from the strain using a chromatography method. Compound 3 showed the highest ADA inhibitory activity and was identified as 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, based on the results of 1H and 13C NMR spectroscopy. The results of molecular docking suggested that compound 3 binds to the active site and the nonspecific binding site of the ADA. Furthermore, we found that compound 3 is a mixed ADA inhibitor. These results indicate that endophytic strains are a promising source of ADA inhibitors and that compound 3 may be a superior source for use in the preparation of biologically active ADA inhibitor compounds used to treat cancer.

Hydrophilic and Insoluble Electrospun Cellulose Acetate Fiber-Based Biosensing Platform for 25-Hydroxy Vitamin-D3 Detection

The present work describes the fabrication of a simple, low-cost, eco-friendly, and disposable conducting paper substrate (RCP) decorated with electrospun cellulose acetate fibers (CAEFs) for biosensor application. CAEFs were electrospun under spinning conditions at a voltage of 16 kV, flow rate of 0.2 mL h–1, and tip-to-collector distance of 15 cm. CAEFs were directly collected onto RCP. A scanning electron microscopy study revealed the diameter of CAEFs in the range of 330 ± 3.50 nm. The contact angle of CAEFs was found to be 38.07 ± 5.10°, which indicates a hydrophilic nature of the fibers. Further, the CAEF/RCP electrode was modified with antibody specific to 25-hydroxy vitamin-D3 (AB-25OHD3), followed with bovine serum albumin (BSA) to block nonspecific binding sites for detection of 25-OH vitamin-D3. The BSA/AB-25OHD3/CAEF/RCP immunoelectrode fabrication was confirmed thorough scanning electron microscopy, Fourier transform infrared spectroscopy, contact angle and electrochemical techniques. The chr...

COUP-TFII is a modulator of cell-type-specific genetic programs based on genomic localization maps

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is a member of the steroid/thyroid hormone receptor superfamily, but its ligand has not yet been identified. Little is known about the role of the COUP-TFII nuclear receptor in cancer cells. In this study, we mapped the cistrome of COUP-TFII in three different cancer cells, namely breast cancer cells (MCF-7), myelogenous leukaemia cells (K562) and liver cancer cells (HepG2) using publicly available ChIP-seq data. Our results show that COUP-TFII co-localises with master transcription factors (TFs) in a cell-specific manner such as estrogen receptor alpha in MCF-7, hepatocyte nuclear factor alpha in HepG2, and GATA-binding factor in K562, while the shared, non-specific COUP-TFII binding sites are co-occupied by CTCF. We identified chromatin environments for these COUP-TFII and master TF co-bound sites together with COUP-TFII and CTCF co-bound sites. Our results show that COUP-TFII and master TF co-bound sites are marked with active enhancer specific histone modifications (H3K27ac and H3K4me1), while COUP-TFII and CTCF co-bound sites reveal active promoter specific histone marks (H3K27ac and H3K4me3). These results describe the genomic context and role of COUP-TFII in the cell-type specific transcriptional programs. Furthermore, we report that the VEGFA gene regulated by shared COUP-TFII and CTCF co-bound regulatory elements is involved in long-range looping in a cell-type-independent manner. These findings provide a genomic insight into the regulation and angiogenic role of COUP-TFII.

A conserved role for transcription factor sumoylation in binding-site selection.

Large numbers of eukaryotic transcription factors (TFs) are modified by SUMO post-translational modifications. Whereas the effect of TF sumoylation on the expression of target genes is largely context-dependent, it is not known whether the modification has a common function in regulating TFs throughout eukaryotic species. Here, I highlight four studies that used genome-wide chromatin-immunoprecipitation analysis (ChIP-seq) to examine whether sumoylation affects the selection of sites on the genome that are bound by human and yeast TFs. The studies found that impairing sumoylation led to deregulated binding-site selection for all four of the examined TFs. Predominantly, compared to wild-type forms, the sumoylation-deficient forms of the TFs bound to numerous additional non-specific sites, pointing to a common role for the modification in restricting TF binding to appropriate sites. Evidence from these studies suggests that TF sumoylation influences binding-site selection by modulating protein-protein interactions with other DNA-binding TFs, or by promoting conformational changes in the TFs that alter their DNA-binding specificity or affinity. I propose a model in which, prior to their sumoylation, TFs initially bind to chromatin with reduced specificity, which leads to spurious binding but also ensures that all functional sites become bound. Once the TFs are bound to DNA, sumoylation then acts to increase specificity and promotes release of the TFs from non-specific sites. The similar observations from these four genome-wide studies across divergent species suggest that binding-site selection is a general and conserved function for TF sumoylation.

A Label-free Cardiac Troponin T Electrochemiluminescence Immunosensor Enhanced by Graphene Nanoplatelets.

In this study, a direct and label-free immunosensor was designed and constructed by modifying the screen-printed electrode with graphene nanoplatelets (GNPs) for the detection of the cardiac troponin T (cTnT). Firstly, GNPs were drop-casted onto carbon working electrode. Monoclonal cTnT antibodies were then immobilized on the GNPs via physical adsorption; finally, BSA was introduced to block non-specific binding sites. The detection of cTnT was performed using an electrochemiluminescence (ECL) technique with tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) used as a luminophore and TPrA (tripropylamine) as a co-reactant. The ECL intensity was demonstrated to be directly proportional to the cTnT concentration where a linear range from 100 pg mL-1 to 5 fg mL-1 of the cTnT detection was established. An extremely low limit of detection was achieved to be 0.05 fg mL-1 with an outstanding specificity. Additionally, this immunosensor showed excellent percentage recovery for real samples analyses in artificially spiked human serum.

Construction of ultrasensitive label-free aptasensor for thrombin detection using palladium nanocones boosted electrochemiluminescence system

Aptamer-based bioassay of biomarker has broad applications in clinical research and disease diagnosis. In this work, a label-free aptamer biosensor for ultrasensitive thrombin (TB) detection was constructed based on highly enhanced electrochemiluminescence (ECL) of functional palladium nanocones (Pd NCs) with tripropylamine (TPA) system. Herein, well-defined Pd NCs were initially functionalized by mercaptoethanol (MCH) as an ECL emitter and immobilized onto the glassy carbon electrode (GCE) initially grafted with amine-terminated polyamidoamine (PAMAM) via electro-oxidation reaction. Under optimal conditions, the ECL intensity of the Pd NCs/TPA system was about 2.5 times enhancement when compared to that of bare GCE. The ECL and cyclic voltammetry (CV) were synchronously employed to illustrate the enhanced ECL mechanism. By virtue of the linkage of the modified Pd NCs with specified TB aptamer (TBA) containing sulfydryl group, a label-free ECL detection platform was designed. After full coverage of the nonspecific sites with bovine serum albumin (BSA), an ECL biosensor for TB detection was constructed by taking the advantage of the steric hindrance effects, showing the wider linear range, lower detection limit of TB (even down to 6.76 fM), superb selectivity and stability. The current strategy provides a highly sensitive platform for detection of various biomolecules in bioanalysis.

Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT-DBD interaction.

Bioinspired synthesis of organic–inorganic hybrid nanoflowers for robust enzyme-free electrochemical immunoassay

Protein-inorganic nanoflowers have been extensively used for sensing and biosensing applications by virtue of the signal enhancement of protein component---enzyme. Can the inorganic component of protein-inorganic nanoflowers be employed to amplify signal transducing for enzyme-free detection? In this work, a new kind of BSA-antibodies-copper phosphate hybrid nanoflowers (BSA-Ab2-Cu3(PO4)2) has been prepared by one-pot biomimetic mineralization process as signal enhancer for enzyme-free electrochemical immunoassay. C-reactive protein (CRP) has been chosen as a model biomarker. To the best of our knowledge, it is the first trial using the inorganic component---phosphate ions of BSA-Ab2-Cu3(PO4)2 for transducing electrochemical readout, which features the following advantages: (1) the three-dimensional hierarchical porous nanoflower morphology with a high specific surface area could load more antibodies, and BSA for blocking non-specific sites, greatly increasing the sensitivity of the fabricated immunosensors, (2) the Cu3(PO4)2 hybrid nanoflowers can supply a huge amount of phosphate anions to react with molybdate yielding molybdophosphate precipitates and generating redox currents for more robust enzyme-free electrochemical signal readout. The fabricated immunosensor has exhibited good detection performance with a linear range of 5 pg/mL–1 ng/mL and a limit of detection of 1.26 pg/mL. Moreover, our method has presented good feasibility for clinical sample analysis.

An Exfoliated Graphite-Based Electrochemical Immunosensor on a Dendrimer/Carbon Nanodot Platform for the Detection of Carcinoembryonic Antigen Cancer Biomarker.

An electrochemical immunosensor for the quantification of carcinoembryonic antigen (CEA) using a nanocomposite of polypropylene imine dendrimer (PPI) and carbon nanodots (CNDTs) on an exfoliated graphite electrode (EG) is reported. The carbon nanodots were prepared by pyrolysis of oats. The nanocomposites (PPI and CNDTs) were characterized using X-ray powder diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), high-resolution transmission electron microscopy (HRTEM) and scanning electron microscopy (SEM). The proposed immunosensor was prepared on an exfoliated graphite electrode sequentially by drop coating CNDTs, the electrodeposition of G2-PPI (generation 2 poly (propylene imine) dendrimer), the immobilization of anti-CEA on the modified electrode for 80 min at 35 °C, and dropping of bovine serum albumin (BSA) to minimize non-specific binding sites. Cyclic voltammetry was used to characterize each stage of the fabrication of the immunosensor. The proposed immunosensor detected CEA within a concentration range of 0.005 to 300 ng/mL with a detection limit of 0.00145 ng/mL by using differential pulse voltammetry (DPV). The immunosensor displayed good stability and was also selective in the presence of some interference species such as ascorbic acid, glucose, alpha-fetoprotein, prostate-specific antigen and human immunoglobulin. Furthermore, the fabricated immunosensor was applied in the quantification of CEA in a human serum sample, indicating its potential for real sample analysis.

PAMAM/polyhedral nanogold-modified probes with DNAase catalysis for the amperometric electrochemical detection of metastasis-associated lung adenocarcinoma transcript\xa01

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non coding RNA (lncRNA) present in serum, is an important biomarker for detecting hepatocellular carcinoma (HCC). However, there are some shortcomings in current detection methods. So developing other novel MALAT1 detection methods is necessary. Electrochemical biosensors using different types of nanomaterials with various advantages may provide a suitable method for detection. Here, a new strategy for MALAT1 detection was proposed based on polyhedral nanogold-polyamide-amine dendrimers (PNG-PAMAMs). The SWCNH/Au composite was used as a capture probe immobilization matrix, and PNG-PAMAM was used as a trace label for the detection probe (DP). The strategy takes advantage of the ability of the surface of PNG to bind a capture probe whose sequence contains (GGG)3 trimer that can bind DNAzyme hemin. Moreover, PNG may carry abundant horseradish peroxidases (HRPs) to block excess nonspecific adsorption sites, with synergistic hemin catalysis. The results show that the biosensor provides ultrasensitive detection of MALAT1 with a remarkable catalytic effect. The enhanced biosensor has a detection limit of 0.22 fmol·mL− 1 for MALAT1, and the linear calibration of the biosensor ranged from 1 fmol·mL− 1 to 100 pmol·mL− 1. In addition, the electrochemical biosensor has desirable qualities compared to other detectors; for instance, it is inexpensive, highly stable, and sensitive and has good reproducibility. This assay was also successfully applied to the detection of MALAT1 in serum samples, demonstrating that the technology has potential application in the detection of MALAT1 for clinical HCC diagnosis.Graphical The schematic presentation ilustrates MALATI detection by biosensor with differential pulse stripping voltammetry. Polyhedral nanogold-PAMAM/horseradish peroxidases (PNG-PAMAM/HRP) detection probe with DNAzyme (hemin) sites was applied to determine MALATI. Signal was amplified by hemin/HRP/H2O2 catalytic system.