The ability to detect single protein molecules in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as approximately 10-20 enzyme-labeled complexes in 100 microl of sample (approximately 10(-19) M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10(-15) M) much lower than conventional ELISA. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).

/pdf/single-molecule-enzyme-linked-immunosorbent-assay-detects-3abhpkc0jx.pdf

Single-Molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations

Many human diseases, including metabolic, immune and central nervous system disorders, as well as cancer, are the consequence of an alteration in lipid metabolic enzymes and their pathways. This illustrates the fundamental role played by lipids in maintaining membrane homeostasis and normal function in healthy cells. We reviewed the major lipid dysfunctions occurring during tumor development, as determined using systems biology approaches. In it, we provide detailed insight into the essential roles exerted by specific lipids in mediating intracellular oncogenic signaling, endoplasmic reticulum stress and bidirectional crosstalk between cells of the tumor microenvironment and cancer cells. Finally, we summarize the advances in ongoing research aimed at exploiting the dependency of cancer cells on lipids to abolish tumor progression.

/pdf/lipid-metabolic-reprogramming-in-cancer-cells-3ir4lx50ud.pdf

Lipid metabolic reprogramming in cancer cells

Natural product and natural product derived drugs in clinical trials

A novel electrochemical immunosensor for sensitive detection of cancer biomarker α-fetoprotein (AFP) is described that uses a graphene sheet sensor platform and functionalized carbon nanospheres (CNSs) labeled with horseradish peroxidase-secondary antibodies (HRP-Ab2). Greatly enhanced sensitivity for the cancer biomarker is based on a dual signal amplification strategy: first, the synthesized CNSs yielded a homogeneous and narrow size distribution, which allowed several binding events of HRP-Ab2 on each nanosphere. Enhanced sensitivity was achieved by introducing the multibioconjugates of HRP-Ab2-CNSs onto the electrode surface through “sandwich” immunoreactions. Second, functionalized graphene sheets used for the biosensor platform increased the surface area to capture a large amount of primary antibodies (Ab1), thus amplifying the detection response. On the basis of the dual signal amplification strategy of graphene sheets and the multienzyme labeling, the developed immunosensor showed a 7-fold increas...

/pdf/sensitive-immunosensor-for-cancer-biomarker-based-on-dual-3lh6g6j2sk.pdf

Sensitive Immunosensor for Cancer Biomarker Based on Dual Signal Amplification Strategy of Graphene Sheets and Multienzyme Functionalized Carbon Nanospheres

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full.

https://bpspubs.onlinelibrary.wiley.com/doi/pdf/10.1111/bph.12451

The Concise Guide to PHARMACOLOGY 2013/14: Enzymes

The bioactive lipid sphingosine-1-phosphate (S1P) is involved in multiple cellular signalling systems and has a pivotal role in the control of immune cell trafficking. As such, S1P has been implicated in disorders such as cancer and inflammatory diseases. This Review discusses the ways in which S1P might be therapeutically targeted — for example, via the development of chemical inhibitors that target the generation, transport and degradation of S1P and via the development of specific S1P receptor agonists. We also highlight recent conflicting results observed in preclinical studies targeting S1P and discuss ongoing clinical trials in this field.

/pdf/targeting-the-sphingosine-1-phosphate-axis-in-cancer-87iheacmjm.pdf

Targeting the sphingosine-1-phosphate axis in cancer, inflammation and beyond

We describe a new format for the recently introduced bio bar code technology, which improves the dose response over 10,000-fold and thereby makes this technique analytically useful. Unlike other ultrasensitive protein detection methods, such as immuno-PCR or immuno-RCA, the bio bar code technique does not employ any enzymes to achieve detection limits in the attomolar range. By sandwiching a target between a magnetic bead and an amplifier nanoparticle, a multiplicity of bar code oligonucleotides are released for each captured target analyte. These surrogate bar code targets are then hybridized to microarrays and detected with silver-amplified gold nanoparticle probes. Using PSA detection as a model, we demonstrate a linear dose response over at least 4 orders of magnitude in both target concentration and concomitant signal and a 1000-fold improvement in detection limit compared to the best ELISA system.

Detection of protein analytes via nanoparticle-based bio bar code technology.

Staphylococcus aureus (SA) is one of the most important human pathogens, causing both nosocomial and community-acquired infections (1)(2). The occurrence of methicillin-resistant SA (MRSA) has increased steadily worldwide and now accounts for a substantial portion of all staphylococcal infections in US hospitals (3). To develop preventive measures, a rapid screening method, along with accurate and timely identification of MRSA, is essential. The existing techniques for doing so are either time-intensive (culturing of bacteria on selective media), relatively insensitive (use of latex agglutination), or expensive and easily susceptible to operator error (such as PCR).

We describe a method designed for clinical laboratories using oligonucleotides conjugated to gold nanoparticles. We avoid radioactivity, fluorescence, or target amplification (such as PCR), and use a simple and rapid hybridization-based approach in a microarray format, with ClearRead™ technology to detect specific genomic sequences (4).

The ClearRead procedure involves a two-step process: the first involves the hybridization of target to oligonucleotides conjugated to gold nanoparticles as well as oligonucleotides attached to a solid matrix; the second step involves the catalytic deposition of silver on the gold nanoparticle, providing a sixfold amplification of signal (Fig. 1A⇓ ) (4)(5). The differentiation at isothermal hybridization temperatures is a result of the sharp melting transitions characteristic of nanoparticle probes (5). Previous methods based on this property (6)(7) have required the use of PCR and have not directly assayed for genomic DNA. Our assay, on the other hand, requires minimal amounts of genomic DNA (∼500 ng, or ∼108 DNA molecules) and has been used to reliably identify MRSA from liquid cultures, based on the detection of the mecA and tuf genes. Resistance to methicillin is mediated by the presence of penicillin-binding protein 2a, encoded by the mecA gene (8)(9). We are …

/pdf/sensitive-assay-for-identification-of-methicillin-resistant-4o03fpf4ml.pdf

Sensitive Assay for Identification of Methicillin-Resistant Staphylococcus aureus, Based on Direct Detection of Genomic DNA by Use of Gold Nanoparticle Probes

Gregory T. Kunkel

Papers

Targeting the sphingosine-1-phosphate axis in cancer, inflammation and beyond

Detection of protein analytes via nanoparticle-based bio bar code technology.

Sensitive Assay for Identification of Methicillin-Resistant Staphylococcus aureus, Based on Direct Detection of Genomic DNA by Use of Gold Nanoparticle Probes

Sam68 Guest STARs in TNF-α Signaling