Streptavidin Biotin Research Articles

Distribution and Appearance of Ki-67, IL-1α, IL-10, and PGP 9.5 in Reinke's Oedema-Affected Larynx Tissue Compared with Control Tissue.

Smoking, laryngopharyngeal reflux, and vocal fold abuse can promote the development of Reinke’s oedema, leading to vocal fold dysfunction and injury. The aim of the work was to investigate the appearance and distribution of proliferation marker Ki-67 (Ki-67), interleukin 10 (IL-10), interleukin 1 alpha (IL-1α), and protein gene peptide 9.5 (PGP 9.5) in Reinke’s oedema-affected larynx tissue. Methods: A routine histological and immunohistochemical Reinke’s oedema and control group patient analysis was conducted. We used the biotin–streptavidin biochemical method to detect Ki-67, IL-10, IL-1α, and PGP 9.5 The semiquantitative grading method was used to evaluate immunoreactive cells’ appearance and local distribution. A Mann–Whitney U test and Spearman’s rank coefficient were performed. Results: A low positive correlation between IL-1α epithelial and subepithelial immunoreactive cells in the patient group was found. Mann–Whitney U tests revealed significant patient and control group immunoreactive marker differences. All examined markers showed a higher number of immunoreactive structures in the patient group. Conclusions: Intensive proliferation of the surface epithelium was observed in patient tissues. The notable increase in IL-10 positive structures indicates the dominant anti-inflammatory tissue response. An increased number of IL-1α structures in the larynx epithelium and subepithelium in the patient group is linked to inflammation, proliferation, and tissue remodelling. The PGP 9.5 expression increase is involved in the morphopathogenesis of Reinke’s oedema.

Fluorophore-gold nanoparticle FRET/plasmonic lasers with the streptavidin-biotin complex as the acceptor–donor linkage

Several Förster resonance energy transfer (FRET) lasers have been realized by employing the robust and versatile streptavidin-biotin (SPB) biocomplex as the acceptor–donor linkage. SPB offers a fixed acceptor–donor separation (“ruler”) of <6 nm, which lies within the Förster radius for a broad range of donors and acceptors. A Cy3-SPB-Cy5 conjugate laser (where Cy3 and Cy5 are cyanine dyes) peaking at λ ∼ 708 nm has been observed, and its bandwidth and threshold pump energy (at 532 nm) have been measured to be ∼4.5 nm and 118 µJ (corresponding to a pump energy density of 179 ± 5 µJ/mm2), respectively. Depolarization of the linearly polarized pump optical field by this FRET process is found to be <12%. To tether the acceptor and donor, the SPB complex requires only that either be conjugated, thereby allowing FRET processes to be examined among an extensive set of biomolecules, inorganics, and nanoantenna acceptors, for example. As a result, fluorophore-nanoparticle lasers having characteristics of both FRET lasers and plasmonic emitters have been demonstrated. Laser spectra and the phase shift induced by a 10 or 100 nm gold nanoparticle tethered to the Cy3-SPB complex suggest that both the fluorescent protein and nanoparticle are able to act as an acceptor. The brightness associated with this new class of fluorophore/nanostructure FRET lasers will broaden the scope of accessible biomedical diagnostics, including cellular imaging and the detection of DNA and proteins.

Precise identification of O-linked β-N-acetylglucosamine peptides based on O-mesitylenesulfonylhydroxylamine elimination reaction

氧连接氮乙酰葡萄糖胺(O-GlcNAc)是一种重要的蛋白质翻译后修饰,它在维持机体正常的生命活动中发挥着重要作用。许多研究证实,O-GlcNAc糖基化修饰稳态的破坏与人类多种疾病的发生相关,大规模富集鉴定O-GlcNAc糖基化修饰蛋白有助于发现新的临床疾病诊断标志物。由于O-GlcNAc糖基化修饰丰度较低,形成的糖苷键不稳定,O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定面临一定挑战。近年来,全乙酰化的非天然糖代谢标记技术被广泛应用于O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。然而,最新的研究发现,在细胞代谢标记过程中,全乙酰化的非天然单糖会同时标记半胱氨酸的巯基而引入半胱氨酸巯基-叠氮糖人为修饰物。该副反应在一定程度上干扰了O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。鉴于此,研究发展了一种通过三甲基苯磺酰羟胺(MSH)特异性氧化消除半胱氨酸巯基-叠氮糖人为修饰物的方法,进而显著提高O-GlcNAc糖基化修饰肽段的精准鉴定。该方法建立于温和的磷酸钠缓冲液(50 mmol/L, pH=8)体系,利用过量的MSH,于95 ℃避光振荡反应30 min,可完全消除半胱氨酸巯基-叠氮糖人为修饰物。该方法应用于Hela细胞中,可有效消除叠氮全乙酰化半乳糖胺(Ac4GalNAz)代谢产生的半胱氨酸巯基-叠氮糖人为修饰物,从而成功富集鉴定到157条O-GlcNAc糖基化修饰肽段,归属于130个蛋白质。该方法有效去除了半胱氨酸巯基-叠氮糖人为修饰物对代谢标记结果的干扰,为非天然糖代谢标记技术在糖蛋白组学分析中的应用提供了新的研究策略。

Equipment-free, salt-mediated immobilization of nucleic acids for nucleic acid lateral flow assays

As the world has been facing several deadly virus crises, including Zika virus disease, Ebola virus disease, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19), lateral flow assays (LFAs), which require minimal equipment for point-of-care of viral infectious diseases, are garnering much attention. Accordingly, there is an increasing demand to reduce the time and cost required for manufacturing LFAs. The current study introduces an equipment-free method of salt-mediated immobilization of nucleic acids (SAIoNs) for LFAs. Compared to general DNA immobilization methods such as streptavidin–biotin, UV-irradiation, and heat treatment, our method does not require special equipment (e.g., centrifuge, UV-crosslinker, heating device); therefore, it can be applied in a resource-limited environment with reduced production costs. The immobilization process was streamlined and completed within 30 min. Our method improved the color intensity signal approximately 14 times compared to the method without using SAIoNs and exhibited reproducibility with the long-term storage stability. The proposed method can be used to detect practical targets (e.g., SARS-CoV-2) and facilitates highly sensitive and selective detection of target nucleic acids with multiplexing capability and without any cross-reactivity. This novel immobilization strategy provides a basis for easily and inexpensively developing nucleic acid LFAs combined with various types of nucleic acid amplification.

Morphological Description of Den-3 Virus Infection Cells through EDTA Blood Feed In Aedes Aegypti Mosquitoe

Mosquito Ae. aegypti is the main dengue virus vector which causes the virus to grow and develop properly. The Artificial Membrane Feeding (AMF) method is a method of indirectly transmitting the dengue virus in the Ae. aegypti. This method uses blood feed with EDTA anticoagulant and DEN-3 virus in the insectarium. One way to detect the dengue virus found in Ae mosquito cells. aegypti is an immunohistochemical Streptavidin Biotin Peroxidase Complex (SBPC). In this method, the morphological picture of DEN-3 virus infection cells will be seen through the heparin anticoagulant blood feed using the AMF method. Anticoagulants function to slow the blood clotting to feed the Ae mosquitoes. Aegypti. The aim of this study was to determine the Positive Infection Rate of DEN-3 virus and the morphological picture of DEN-3 virus infection cells using the AMF method of EDTA anticoagulant blood feed through SBPC immunocytochemical examination of Ae mosquitoes. aegypti. The design method of this study was experimental, infected Ae.aegypti mosquitoes using anticoagulant blood containing DEN-3 virus orally as an infectious sample. Negative control used Culex Sp mosquitoes and positive control used adult Ae.aegypti mosquitoes which were infected with DEN-3 virus by injection. Detection of DEN-3 virus in Ae.aegypti mosquitoes through SBPC immunositochemical examination. Results of Positive Infection Rate for Ae.aegypti mosquitoes with EDTA anticoagulant as much as 23.20% through SBPC immunocytochemical examination with a picture of cell morphology of DEN-3 virus infection. The conclusion of SBPC immunositochemical examination showed positive cell morphology of DEN-3 virus infection with EDTA anticoagulant in Ae.aegypti mosquitoes fed by human blood orally through the AMF method.

DNAzyme-Based Biosensors: Immobilization Strategies, Applications, and Future Prospective.

Since their discovery almost three decades ago, DNAzymes have been used extensively in biosensing. Depending on the type of DNAzyme being used, these functional oligonucleotides can act as molecular recognition elements within biosensors, offering high specificity to their target analyte, or as reporters capable of transducing a detectable signal. Several parameters need to be considered when designing a DNAzyme-based biosensor. In particular, given that many of these biosensors immobilize DNAzymes onto a sensing surface, selecting an appropriate immobilization strategy is vital. Suboptimal immobilization can result in both DNAzyme detachment and poor accessibility toward the target, leading to low sensing accuracy and sensitivity. Various approaches have been employed for DNAzyme immobilization within biosensors, ranging from amine and thiol-based covalent attachment to non-covalent strategies involving biotin-streptavidin interactions, DNA hybridization, electrostatic interactions, and physical entrapment. While the properties of each strategy inform its applicability within a proposed sensor, the selection of an appropriate strategy is largely dependent on the desired application. This is especially true given the diverse use of DNAzyme-based biosensors for the detection of pathogens, metal ions, and clinical biomarkers. In an effort to make the development of such sensors easier to navigate, this paper provides a comprehensive review of existing immobilization strategies, with a focus on their respective advantages, drawbacks, and optimal conditions for use. Next, common applications of existing DNAzyme-based biosensors are discussed. Last, emerging and future trends in the development of DNAzyme-based biosensors are discussed, and gaps in existing research worthy of exploration are identified.

Heparin–Avastin Complexes Show Enhanced VEGF Binding and Inhibition of VEGF-Mediated Cell Migration

Bevacizumab (known by the tradename Avastin) is an antibody that binds VEGF and blocks its binding to VEGF receptors on endothelial cells, and is used to treat cancers and other diseases associated with excessive vascular growth. Our previous findings showed enhanced VEGF binding to Avastin in the presence of heparin, indicating that colocalizing heparin with Avastin could enhance VEGF inhibitory activity. Thus, the aim of the present study was to determine if conjugating Avastin and heparin to one another would lead to enhanced anti-VEGF activity. Avastin was conjugated to either biotin or streptavidin, and biotin–heparin was used to bring the two molecules into close proximity via biotin–streptavidin binding. Radioligand binding assays with 125 I-VEGF and cell migration assays using human umbilical vein endothelial cells were used to evaluate the impact of heparin on Avastin binding and activity. We found that bringing Avastin and heparin together, either on a surface or through streptavidin conjugation of Avastin, led to increased VEGF binding compared to that with each molecule alone. The heparin-mediated increase in VEGF binding was also noted at acidic pH where Avastin showed decreased VEGF binding. Conditions where Avastin and heparin showed enhanced VEGF binding also showed reduced VEGF-induced migration of human umbilical vein endothelial cells. These findings suggest design principles for a modified Avastin-based inhibitor of angiogenesis.

Construction of a Dye-Sensitized and Gold Plasmon-Enhanced Cathodic Photoelectrochemical Biosensor for Methyltransferase Activity Assay.

DNA methyltransferases may function as important biomarkers of cancers and genetic diseases. Herein, we develop a dye-sensitized and gold plasmon-enhanced cathodic photoelectrochemical (PEC) biosensor on the basis of p-type covalent organic polymers (COPs) for the signal-on measurement of M.SssI methyltransferase (M.SssI MTase). The cathodic PEC biosensor is constructed by the in situ growth of p-type COP films onto a glass coated with indium tin oxide and the subsequent assembly of biotin- and HS-labeled double-stranded DNA (dsDNA) probes onto the COP film via biotin-streptavidin interaction. The dsDNA probe contains the recognition sequence of M.SssI MTase. The COP thin films possess a porous ultrathin nanosheet structure with abundant active sites, facilitating the generation of a high photocurrent compared with the hydrothermally synthesized ones. The presence of DNA methyltransferases can prevent the digestion of restriction endonuclease HpaII, consequently inducing the introduction of gold nanoparticles (AuNPs) to the dsDNA probes via the S-Au bond and the intercalation of rhodamine B (RhB) into the DNA grooves to produce a high photocurrent due to the dye-photosensitized enhancement and AuNP-mediated surface plasmon resonance. However, in the absence of M.SssI MTase, HpaII digests the dsDNA probes, and neither AuNPs nor RhB can be introduced onto the electrode surface, leading to a low photocurrent. This cathodic PEC biosensor possesses high sensitivity and good selectivity, and it can screen the inhibitors and detect M.SssI MTase in serum as well.

Increased Retention of Cardiac Cells to a Glass Substrate through Streptavidin-Biotin Affinity.

In vitro analysis of primary isolated adult cardiomyocyte physiological processes often involves optical imaging of dye-loaded cells on a glass substrate. However, when exposed to rapid solution changes, primary cardiomyocytes often move to compromise quantitative measures. Improved immobilization of cells to glass would permit higher throughput assays. Here, we engineer the peripheral membrane of cardiomyocytes with biotin to anchor cardiomyocytes to borosilicate glass coverslips functionalized with streptavidin. We use a rat cardiac myoblast cell line to determine general relationships between processing conditions, ligand density on the cell and the glass substrate, cellular function, and cell retention under shear flow. Use of the streptavidin–biotin system allows for more than 80% retention of cardiac myoblasts under conventional rinsing procedures, while unmodified cells are largely rinsed away. The adhesion system enables the in-field retention of cardiac cells during rapid fluid changes using traditional pipetting or a modern microfluidic system at a flow rate of 160 mL/min. Under fluid flow, the surface-engineered primary adult cardiomyocytes are retained in the field of view of the microscope, while unmodified cells are rinsed away. Importantly, the engineered cardiomyocytes are functional following adhesion to the glass substrate, where contractions are readily observed. When applying this adhesion system to cardiomyocyte functional analysis, we measure calcium release transients by caffeine induction at an 80% success rate compared to 20% without surface engineering.

A Rapidly Stabilizing Water-Gated Field-Effect Transistor Based on Printed Single-Walled Carbon Nanotubes for Biosensing Applications.

Biosensors are expected to revolutionize disease management through provision of low-cost diagnostic platforms for molecular and pathogenic detection with high sensitivity and short response time. In this context, there has been an ever-increasing interest in using electrolyte-gated field-effect transistors (EG-FETs) for biosensing applications owing to their expanding potential of being employed for label-free detection of a broad range of biomarkers with high selectivity and sensitivity while operating at sub-volt working potentials. Although organic semiconductors have been widely utilized as the channel in EG-FETs, primarily due to their compatibility with cost-effective low-temperature solution-processing fabrication techniques, alternative carbon-based platforms have the potential to provide similar advantages with improved electronic performances. Here, we propose the use of inkjet-printed polymer-wrapped monochiral single-walled carbon nanotubes (s-SWCNTs) for the channel of EG-FETs in an aqueous environment. In particular, we show that our EG-CNTFETs require only an hour of stabilization before producing a highly stable response suitable for biosensing, with a drastic time reduction with respect to the most exploited organic semiconductor for biosensors. As a proof-of-principle, we successfully employed our water-gated device to detect the well-known biotin–streptavidin binding event.

Novel Bispecific Antibody Targeting CD45 and 90Y-DOTA Is Effective Therapy for Acute Myeloid Leukemia in Preclinical Murine Models

Background : Acute myeloid leukemia (AML) is a heterogeneous disease with a variety of chromosomal and molecular aberrations, rendering AML a challenging disease to cure with targeted agents. Despite the genetic variability, AML uniformly responds to radiation therapy. We have previously shown therapeutic efficacy targeting CD45+ AML using the beta-emitter 90Y and a streptavidin-biotin (SA) pretargeted radioimmunotherapy (PRIT) system. Theoretical concerns, such as endogenous biotin in humans, may limit translation of this approach to the clinic. To circumvent such hurdles, we developed bispecific antibody constructs targeting both CD45 and 90Y-labeled-DOTA-biotin, and evaluated this approach in murine models of AML.Methods : We constructed a pair of plasmids using the pfuse system that code for heavy and light chains for antibody bispecific to murine CD45 (30F11) and Y-DOTA (C825). Plasmid pairs were co-transfected into HEK 293T cells for production and purificaiton of the bispecific antibody. Binding to CD45 and 90Y-labeled-DOTA-biotin was confirmed, after which biodistribution studies with 30F11-Fc-C825 were performed in a disseminated murine leukemia model. B6SJLF1 mice (n=5 per group) were injected intravenously with 105 SJL murine leukemia cells, followed 2 days later with 0.67 nmol 30F11-Fc-C825, then 5 μg DOTA-Y-dextran clearing agent 23 hr later to clear any unbound bispecific construct from circulation. Mice were then injected with 50 µCi 90Y-DOTA-biotin IP 24 hr after the bispecific antibody infusion. Tissues were harvested at 6 and 24 hr after radiolabeled DOTA-biotin, and then analyzed for radioactivity to calculate the percent injected dose per gram of tissue (% ID/g).Results : Target organs (spleen and bone marrow) from leukemia bearing mice treated with 30F11-Fc-C825 had 9.0 ± 1.5 and 8.1 ± 1.2% ID/g, respectively, 24 hr post injection. Non-target organs such as kidneys and lungs each had <0.5% ID/g at this time point. Targeting translated into a therapeutic benefit, as mice bearing SJL leukemia treated with 30F11-Fc-C825 and 800 µCi of 90Y-DOTA-biotin had median overall survival of 43 days, compared to leukemic control mice (30 days, p<0.0001).To investigate this approach in a clinically relevant human leukemia model, an anti-human CD45 (BC8) scFv fusion protein was similarly engineered onto a high-affinity anti-Y-DOTA (C825) scFv fusion protein. This bispecific construct (BC8-Fc-C825) was expressed and purified from CHO cells. Binding to human CD45 and 90Y-labeled-DOTA-biotin was confirmed by flow cytometry. In biodistribution studies, female athymic mice (n=5 per group) were injected subcutaneously with 107 human AML cells (HEL). Mice were then injected intravenously with 1.4 nmol BC8-Fc-C825, BC8-SA, or non-targeting CC49-Fc-C825 as the first step before receiving clearing agent. Radiolabeled 90Y-DOTA-biotin (50 µCi) was injected IP 24 hr after bispecific delivery. HEL tumors from mice treated with BC8-Fc-C825 showed peak uptake at 24 hr post injection with 7.75 ± 0.02% ID/g, with minimal uptake in non-target organs [e.g., 0.5 ± 0.3% ID/g in kidneys]. This compared favorably with HEL tumors from mice treated with BC8-SA as the first step, which had peak uptake of 6.7 ± 0.01% ID/g 24 hr post-injection. Therapy studies took athymic mice (n=10 per group) that had been given subcutaneous HEL xenografts, before receiving BC8-Fc-C825, BC8-SA, or non-binding control CC49-Fc-C825. Treated mice were given clearing agent, followed by 800 to 1500 µCi of 90Y-labeled DOTA-biotin. Mice bearing HEL tumors treated with BC8-Fc-C825 followed by 1400 µCi of 90Y-DOTA-biotin, resulted in 6 of 10 mice surviving 170 days post-injection, whereas untreated controls and HEL-bearing mice treated with non-targeting negative control CC49-Fc-C825 and 1400 µCi of 90Y-DOTA-biotin required euthanasia due to tumor size by day 26 and 32, respectively.Conclusion : Our bispecific constructs both showed selectivity for CD45+ targets and 90Y-DOTA. In two leukemia models, targeting of 90Y-DOTA to target hematopoietic tissues translated into survival benefits. Given the therapeutic efficacy, bispecific constructs with the capacity to pretarget CD45 and capture 90Y-DOTA should be added to the armamentarium of PRIT approaches. [Display omitted] DisclosuresOrozco:Actinium Pharmaceuticals: Other: Research Funding to Institution for sponsored Clinical Trials. Till:Mustang Bio: Patents & Royalties, Research Funding; Genentech: Research Funding. Gopal:Seattle Genetics: Consultancy, Research Funding. Orcutt:inviCRO: Employment, Equity Ownership. Pagel:Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Press:Roche: Honoraria, Research Funding; BMS: Honoraria; Bayer: Consultancy.

Glut 1, S-100, and Nerve Bundle Study in Vascular Anomalies

Vascular anomalies grouped into vascular tumors (hemangioma) (HI) and vascular malformation (VM) are benign vascular lesions that are difficult to distinguish from one another clinically and often confused with each other at histopathology. This confusing terminology leads to improper diagnosis, illogical treatment, and misdirected research. This study aimed to study GLUT 1, S-100, and nerve bundle to differentiate hemangioma and vascular malformation. Thirty two cases of vascular lesions (26 vascular malformations and 6 hemangiomas) were taken into the study. For histological evaluation and immunohistochemistry (IHC), samples of vascular lesions were collected in formalin. All the hematoxylin and eosin-stained slides were evaluated under light microscope for histology and nerve bundles. Immunohistochemical staining was performed by streptavidin–biotin method for GLUT 1 and S-100. GLUT 1 was positive in all 6 cases of hemangiomas (100%) and only in 1 case of vascular malformation. Nerve bundle was present in 24 cases of vascular malformation (92.3%) out of 26 cases but not in any cases of hemangioma and S-100 was found positive in all vascular malformation cases (100%) but not in hemangioma. So GLUT 1 expression, S-100, and presence of nerve bundle in vascular lesions can help to differentiate hemangioma and vascular malformation.

Electrochemical Biosensor with Enhanced Antifouling Capability for COVID-19 Nucleic Acid Detection in Complex Biological Media.

Biofouling caused by the accumulation of biomolecules on sensing surfaces is one of the major problems and challenges to realize the practical application of electrochemical biosensors, and an effective way to counter this problem is the construction of antifouling biosensors. Herein, an antifouling electrochemical biosensor was constructed based on electropolymerized polyaniline (PANI) nanowires and newly designed peptides for the detection of the COVID-19 N-gene. The inverted Y-shaped peptides were designed with excellent antifouling properties and two anchoring branches, and their antifouling performances against proteins and complex biological media were investigated using different approaches. Based on the biotin–streptavidin affinity system, biotin-labeled probes specific to the N-gene (nucleocapsid phosphoprotein) of COVID-19 were immobilized onto the peptide-coated PANI nanowires, forming a highly sensitive and antifouling electrochemical sensing interface for the detection of COVID-19 nucleic acid. The antifouling genosensor demonstrated a wide linear range (10–14 to 10–9 M) and an exceptional low detection limit (3.5 fM). The remarkable performance of the genosensor derives from the high peak current of PANI, which is chosen as the sensing signal, and the extraordinary antifouling properties of designed peptides, which guarantee accurate detection in complex systems. These crucial features represent essential elements for future rapid and decentralized clinical testing.

Succinimidyl Carbonate-Based Amine-Reactive Polymer Brushes: Facile Fabrication of Functional Interfaces

Polymeric coatings that can undergo effective functionalization with ligands and (bio)molecules necessary for intended applications are widely employed to engineer functional interfaces. Herein, amine-reactive polymer brushes are fabricated using a succinimidyl group-activated carbonate monomer, and their facile post-polymerization functionalization is demonstrated through ligand-mediated protein immobilization and detection. Copolymer brushes containing varying amounts of the reactive monomer and an ethylene glycol-based methacrylate comonomer were obtained using surface-mediated reversible addition fragmentation chain transfer polymerization. Post-polymerization functionalization of the thus-obtained copolymer brushes with a fluorine-containing amine, namely, 4-(trifluoromethyl)benzylamine, demonstrated highly efficient functionalization at ambient temperature. To demonstrate possible applications, polymer brushes functionalized with a bioactive ligand, namely, biotin, were used to detect the target protein, streptavidin. The biotin–streptavidin interaction on brushes could also be employed to conjugate protein-coated quantum dots. Importantly, comparison of the attachment of 4-(trifluoromethyl)benzylamine on activated carbonate group-containing brushes with reaction of the same molecule on traditional active ester-based brushes demonstrated higher extent of conjugation to carbonate brushes. Finally, orthogonal functionalization of the copolymer brushes with an amino-functionalized molecule and a maleimide-containing fluorescent dye in spatial control using microcontact printing was demonstrated. One can envision that the facile fabrication and efficient functionalization of succinimidyl carbonate-based amine-reactive brushes afford an attractive platform for applications using functional interfaces for diagnostic purposes.

Relations of Human Epidermal Growth Factor Receptor 2 (HER2) Expression and E-Cadherin (CDH1) Expression in Breast Cancer Patients

The aim of this study is to analyze the relations of Human Epidermal Growth Factor Receptor 2 (HER2) expression and E-Cadherin (CDH1) expression in breast cancer patients. To date, the synergistic effect of this CDH1/HER2 complex is not well clarified. The design of this study was cross-sectional with a total sample of 56 formalin-fixed paraffin tissue blocks that had been examined for HER2. Furthermore, CDHI expression was examined using the Immunohistochemistry staining technique with the Labeled Streptavidin Biotin Complex (LSAB) method. Bivariate analysis was performed using the Spearman correlation test with abnormally distributed data (p&gt;0.05). Of the 56 data on breast cancer patients, most of the patients (87.5%) were diagnosed at the age of ≥40 years. The majority of cancer staging was IIIB, which was 42.9% of the total 56 patients. The study results shows that 80.0% of HER2-positive patients were in the strong CDH1 group. From these data, there is evidence of correlation between HER2 expression and CDH1 expression in breast cancer patients, however this correlation was not significant (p&gt;0.05).

Specific detection of Staphylococcus aureus infection and marker for Alzheimer disease by surface enhanced Raman spectroscopy using silver and gold nanoparticle-coated magnetic polystyrene beads

Targeted and effective therapy of diseases demands utilization of rapid methods of identification of the given markers. Surface enhanced Raman spectroscopy (SERS) in conjunction with streptavidin–biotin complex is a promising alternative to culture or PCR based methods used for such purposes. Many biotinylated antibodies are available on the market and so this system offers a powerful tool for many analytical applications. Here, we present a very fast and easy-to-use procedure for preparation of streptavidin coated magnetic polystyrene–Au (or Ag) nanocomposite particles as efficient substrate for surface SERS purposes. As a precursor for the preparation of SERS active and magnetically separable composite, commercially available streptavidin coated polystyrene (PS) microparticles with a magnetic core were utilized. These composites of PS particles with silver or gold nanoparticles were prepared by reducing Au(III) or Ag(I) ions using ascorbic acid or dopamine. The choice of the reducing agent influences the morphology and the size of the prepared Ag or Au particles (15–100 nm). The prepare composites were also characterized by HR-TEM images, mapping of elements and also magnetization measurements. The content of Au and Ag was determined by AAS analysis. The synthesized composites have a significantly lower density against magnetic composites based on iron oxides, which considerably decreases the tendency to sedimentation. The polystyrene shell on a magnetic iron oxide core also pronouncedly reduces the inclination to particle aggregation. Moreover, the preparation and purification of this SERS substrate takes only a few minutes. The PS composite with thorny Au particles with the size of approximately 100 nm prepared was utilized for specific and selective detection of Staphylococcus aureus infection in joint knee fluid (PJI) and tau protein (marker for Alzheimer disease).

Reusable pectin‐coated magnetic nanosorbent functionalized with an aptamer for highly selective Hg2+ detection

AbstractA pectin‐coated magnetite nanoparticle functionalized with Hg2+‐specific aptamer was developed to obtain a magnetic nanosorbent having high selectivity toward Hg2+ and facile reusability. The aptamer labeled with biotin was immobilized on pectin‐coated MNP conjugated with streptavidin through the specific biotin–streptavidin interaction to obtain an aptamer‐functionalized nanosorbent. Pectin coated on the particle improved its stability and dispersibility in water, while thymine in the aptamer played an important role as a recognition element, which can bind to the Hg2+ target via T–Hg2+–T complexes. The nanosorbent showed a linear concentration, ranging from 2.8 to 700 μg/L, with the limit of detection of 0.8 ± 0.2 μg/L and limit of quantitation of 2.8 ± 0.2 μg/L for the detection of Hg2+. It showed high selectivity with good tolerance toward Hg2+ in the presence of other metal ions. The system can be repeatedly used for up to 10 cycles of Hg2+ adsorption with only slight changes in its performance. In addition, satisfactory recoveries were also obtained for the detection of Hg2+ in real water samples. This nanosorbent might be a promising tool for extended applications in biological and analytical fields.

DNA-based functionalization of two-dimensional MoS2 FET biosensor for ultrasensitive detection of PSA

Molybdenum disulfide (MoS2) field-effect transistor (FET) biosensors are being increasingly studied due to their highly sensitive and label-free biodetection, system-level integration and easy fabrication. However, the stability and detection ability of a pristine device deteriorate due to the absorption of H2O and CO and usually exhibit high false signals. Herein, a novel ecofriendly technology is employed to functionalize MoS2 FETs using the conjugation of a DNA tetrahedron and biotin-streptavidin (B-SA). The functionlized devices demonstrate ultrahigh sensitivity of the target protein (prostate-specific antigen, PSA) with a limit of detection of 1fg/mL and a wide linear range of 1fg/mL to 100ng/mL in a phosphate-buffered saline, which are 100 and 10,000 times higher than the reported highest values, respectively; these results are attributed to the stable immobilization provided by the DNA tetrahedron as well as the multilevel amplification and anchor system (B-SA system), which improves the antibody-antigen (Ab-Ag) binding. Real-time measurement results indicate that our proposed biosensor can also detect trace PSA (1fg/mL) in a clinically relevant sample of healthy human serum.

An aptasensor using ceria electrodeposited-screen-printed carbon electrode for detection of epithelial sodium channel protein as a hypertension biomarker.

Epithelial sodium channel (ENaC) is a transmembrane protein that has an essential role in maintaining the levels of sodium in blood plasma. A person with a family history of hypertension has a high enough amount of ENaC protein in the kidneys or other organs, so that the ENaC protein acts as a marker that a person is susceptible to hypertension. An aptasensor involves aptamers, which are oligonucleotides that function similar to antibodies, as sensing elements. An electrochemical aptasensor for the detection of ENaC was developed using a screen-printed carbon electrode (SPCE) which was modified by electrodeposition of cerium oxide (CeO2). The aptamer immobilization was via the streptavidin–biotin system. The measurement of changes in current of the active redox [Fe(CN)6]3−/4− was carried out by differential pulse voltammetry. The surfaces of SPCE and SPCE/CeO2 were characterized using scanning electron microscopy, voltammetry and electrochemical impedance spectroscopy. The Box–Behnken experimental optimization design revealed the streptavidin incubation time, aptamer incubation time and streptavidin concentrations were 30 min, 30 min and 10.8 µg ml−1, respectively. Various concentrations of ENaC were used to obtain the linearity range of 0.05–3.0 ng ml−1, and the limits of detection and quantification were 0.012 ng ml−1 and 0.038 ng ml−1, respectively. This aptasensor method has the potential to measure the ENaC protein levels in urine samples as well as to be a point-of-care device.

Protease Biosensor by Conversion of a Homogeneous Assay into a Surface-Tethered Electrochemical Analysis Based on Streptavidin-Biotin Interactions.

This work proposed a new sensing strategy for protease detection by converting a homogeneous assay into a surface-tethered electrochemical analysis. Streptavidin (SA), a tetramer protein, was used as the sensing unit based on the SA-biotin coupling chemistry. Caspase-3 was used as the model analyte, and a biotinylated peptide with a sequence of biotin-GDEVDGK-biotin was designed as the substrate. Specifically, the peptide substrate could induce an assembly of SA to form (SA-biotin-GDEVDGK-biotin)n aggregates through SA-biotin interactions, which was confirmed by atomic force microscopy (AFM). The peptide substrate-induced assembly of SA was facilely initiated on an electrode-liquid surface by modification of the electrode with SA. The in situ formation of (SA-biotin-GDEVDGK-biotin)n aggregates created an insulating layer, thus limiting the electron transfer of ferricyanide. Once the peptide substrate was cleaved into two shorter fragments (biotin-GDEVD and GK-biotin) by caspase-3, the resulting products would compete with biotin-GDEVDGK-biotin to bind SA proteins immobilized on the electrode surface and distributed in a solution, thus preventing the in situ formation of (SA-biotin-GDEVDGK-biotin)n assemblies. With the simple principle of the substrate-induced assembly of SA, a dual-signal amplification was achieved with improved sensitivity. Taking advantage of high sensitivity, simple principle, and easy operation, this method can be augmented to design various surface-tethered biosensors for practical applications.