Rapid integrated biosensor for multiplexed immunoassays based on actuated magnetic nanoparticles.

The realization of biomolecular detection assays for diagnostic purposes is technologically very challenging because such tests demand full integration for ease of use and need to deliver a high analytical performance with cost-effective use of materials. In this article an optomagnetic immunoassay technology is described based on nanoparticles that are magnetically actuated and optically detected in a stationary sample fluid. The dynamic control of nanoparticles by magnetic fields impacts the key immunoassay process steps, giving unprecedented speed, assay control and seamless integration of the total test. The optical detection yields sensitive and multiplexed assays in a low-cost disposable cartridge. We demonstrate that the optomagnetic technology enables high-sensitivity one-step assays in blood serum/plasma and whole saliva. Drugs of abuse are detected at sub-nanogram per millilitre levels in a total assay time of 1 min, and the cardiac marker troponin I is detected at sub-picomole per litre concentrations in a few minutes. The optomagnetic technology is fundamentally suited for high-performance integrated testing and is expected to open a new paradigm in biosensing.

Rapid DNA multi-analyte immunoassay on a magneto-resistance biosensor.

We present the rapid and sensitive detection of amplified DNA on a giant magneto-resistance sensor using superparamagnetic particles as a detection label. The one-step assay is performed on an integrated and miniaturized detection platform suitable for application into point-of-care devices. A double-tagged PCR amplification product of the LamB gene of the Escherichia coli bacterium was used to investigate binding kinetics of the assay. We applied magnetic actuation to concentrate the target-particle complexes at the sensor surface and to remove unbound particles from the sensor surface. We achieved biological dose-response curves detecting 4-250pM amplicon concentrations in a one-step format in total assay times of less than 3min. Using various tag-antibody combinations specific for one of the individual genes, multi-analyte detection is shown of several antibiotic resistance genes of the food pathogen Salmonella.

Osmotic cell swelling-induced ATP release mediates the activation of extracellular signal-regulated protein kinase (Erk)-1/2 but not the activation of osmo-sensitive anion channels.

Human intestine 407 cells respond to hypo-osmotic stress by the rapid release of ATP into the extracellular medium. A difference in the time course of activation as well as in the sensitivity to cytochalasin B treatment and BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester] loading suggests that ATP leaves the cell through a pathway distinct from volume-regulated anion channels. To evaluate a putative role for nucleotides as autocrinic/paracrinic factors in osmotic signalling, the effects of extracellular ATP on the regulation of volume-sensitive anion channels as well as on the hypotonicity-induced activation of extracellular signal-regulated protein kinases (Erk-1/2) were investigated. Micromolar concentrations of ATP were unable to elicit an isotope efflux from (125)I(-)-loaded cells by itself, but strongly potentiated the hypotonicity-provoked anion efflux through a Ca(2+)-dependent mechanism. The order of potency of nucleotides (ATP = UTP = ATP[S] > ADP = AMP >> adenosine = cAMP) indicated the involvement of P2Y(2) receptors. In contrast, millimolar concentrations of ATP markedly inhibited both the osmotically induced isotope efflux and whole-cell Cl(-) currents. Inhibition of whole-cell Cl(-) currents, not only by millimolar ATP but also by the purinoceptor antagonists suramin and reactive blue, was observed most prominently at depolarizing holding potentials, suggesting a direct interaction with volume-sensitive Cl(-) channels rather than interaction with purinoceptors. Both ATP and UTP, at submicromolar levels, were found to act as potent activators of Erk-1/2 in intestine 407 cells. Addition of the ATP hydrolase apyrase to the bath greatly reduced the hypotonicity-induced Erk-1/2 activation, but did not affect the swelling-induced isotope efflux or whole-cell Cl(-) currents. Furthermore, pre-treatment with suramin or reactive blue almost completely prevented the hypo-osmotic activation of Erk-1/2. The results indicate that extracellularly released ATP functions as an autocrinic/paracrinic factor that mediates hypotonicity-induced Erk-1/2 activation but does not serve as an activator of volume-sensitive compensatory Cl(-) currents.

Osmotic swelling-induced activation of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2 in intestine 407 cells involves the Ras/Raf-signalling pathway.

Human Intestine 407 cells respond to hypo-osmotic stress with a rapid stimulation of compensatory ionic conductances accompanied by a transient increase in the activity of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2. In this study, we examined the upstream regulators of hypotonicity-induced Erk-1/Erk-2 activation and their possible role in cell-volume regulation. The hypotonicity-provoked Erk-1/Erk-2 activation was greatly reduced in cells pretreated with the specific mitogen-activated/Erk-activating kinase inhibitor PD098059 and was preceded by a transient stimulation of Raf-1. Pretreatment of the cells with PMA, GF109203X, wortmannin or Clostridium botulinum C3 exoenzyme did not appreciably affect the hypotonicity-provoked Erk-1/Erk-2 stimulation, suggesting the osmosensitive signalling pathway to be largely independent of protein kinase C and p21(rho). In contrast, expression of dominant negative RasN17 completely abolished the hypotonicity-induced Erk-1/Erk-2 activation. Stimulation of the swelling-induced ion efflux was independent of activation of these mitogen-activated protein kinases, as revealed by hypotonicity-provoked isotope efflux from 125I-- and 86Rb+-loaded cells after pretreatment with PD098059 and after expression of RasN17. In addition, the epidermal-growth-factor-induced potentiation of the hypotonicity-provoked anionic response did not depend on the increase in Erk-1/Erk-2 activity but, instead, was found to depend on Ca2+ influx. Taken together, these results indicate that hypotonic stress induces Erk-1/Erk-2 activation through the Ras/Raf-signalling pathway, and argue against a direct role for this pathway in cell-volume control.

Macrophages influence gap junctional intercellular communication between smooth muscle cells in a co-culture model.

The present study demonstrates for the first time that cells cultured on pore membrane inserts (macrophages) modulate gap junctional intercellular communication (GJIC) between a second cell type (smooth muscle cells (SMC)) co-cultured in Transwell-COL cell culture chambers. Unstimulated J774A.1 murine macrophages reduced GJIC between human SMC. Stimulation of J774A.1 cells by lipopolysaccharide (LPS) or interferon-γ abrogated this modulation of GJIC. Unstimulated human monocyte-macrophages did not affect GJIC between human SMC. Upon stimulation of these monocyte-macrophages with LPS, a substantial increase in GJIC between co-cultured SMC was observed. Thus, activation of macrophages alters their interaction with co-cultured SMC. Since these results were obtained in an indirect co-culture system in which direct cell-cell contact is prevented, it is hypothesized that soluble factors released by macrophages may be involved in this modulation of GJIC between SMC. The possible nature of the responsible soluble factors is discussed in the context of atherosclerosis.

Sucrose polyester does not inhibit gastric acid secretion or stimulate cholecystokinin release in men.

Replacement of dietary fat by sucrose polyester reduces fat intake. However, little is known about the effects of sucrose polyester on gastrointestinal function. To investigate the effect on gastric acid secretion and on release of cholecystokinin into plasma, we perfused eight healthy male volunteers intraduodenally with sucrose polyester, digestible fat, or saline on separate days in random order. Intraduodenal perfusion of sucrose polyester did not suppress gastrin-stimulated gastric acid secretion (-1.8 +/- 6.8%) whereas digestible fat suppressed gastric acid secretion by 64 +/- 9% (P = 0.001) compared with saline. Sucrose polyester did not affect plasma cholecystokinin concentrations (-12.8 +/- 9.3 pmol.30 min/L) whereas perfusion with digestible fat resulted in a significant increase (31.7 +/- 9.3 pmol.30 min/L, P = 0.017) compared with saline. We conclude that sucrose polyester, in contrast with digestible fat, does not inhibit gastrin-stimulated gastric acid secretion or stimulate release of cholecystokinin.

Modulation of intercellular communication between smooth muscle cells by growth factors and cytokines.

We recently reported that tumor necrosis factor alpha is able to cause a dose-dependent and persistent reduction in gap junctional intercellular communication between primary human smooth muscle cells. In order to study whether this observed persistent reduction in gap junctional intercellular communication is a unique feature for tumor necrosis factor alpha, the present study focuses on the effects of other growth factors and cytokines on gap junctional intercellular communication. Platelet-derived growth factor AA and BB (PDGF-AA, PDGF-BB), basic fibroblast growth factor (bFGF), interleukin-6 and interferon-gamma were able to modulate gap junctional intercellular communication between primary human smooth muscle cells in vitro. However, our results demonstrate that the magnitude and nature of the observed effects are growth factor- and cytokine-specific. PDGF-AA, PDGF-BB and interleukin-6 caused a transient reduction in gap junctional intercellular communication, while bFGF induced a transient increase in gap junctional intercellular communication. Interferon-gamma was shown to be capable of causing a persistent reduction in gap junctional intercellular communication. In addition, PDGF-AA, PDGF-BB, bFGF, interleukin-6, interferon-gamma and tumor necrosis factor alpha all stimulated smooth muscle cell proliferation. These observations suggest a more complex relationship between modulation of gap junctional intercellular communication and cell proliferation than current hypotheses imply. The implications of the observed effects of growth factors and cytokines on gap junctional intercellular communication between smooth muscle cells in relation to the process of atherosclerosis is discussed.

Determinants of hand function in patients with rheumatoid arthritis.

OBJECTIVE: To investigate which clinical variables contribute to the function of the hand in activities of daily living (ADL) in patients with rheumatoid arthritis (RA). METHODS: In 50 patients with RA hand function in ADL was assessed by (1) the dexterity items of the Arthritis Impact Measurement Scales (AIMS), (2) direct observation of the same items by an occupational therapist, and (3) the Jebsen hand function test. A combined hand function factor was constructed by principal component analysis of the 3 hand function measures. Further assessments included measurements of muscle strength, deformity and destruction, range of motion, and local arthritis activity. After bivariate analyses, variables that correlated significantly with the measures of hand function were entered into stepwise multiple regression analyses. RESULTS: The variables having a significant correlation with most of the hand function measures were: pinch and grip strength, patient's assessment of pain and stiffness of the hands, flexion of the thumb and fingers, range of motion of the wrist, alignment of the metacarpophalangeal (MCP) joints, swan neck and Z deformities, and the Larsen erosion score. 78% of the variance of the combined hand function factor could be explained by pinch strength, stiffness of the hands, and the presence of Z deformity and ulnar deviation. CONCLUSION: Pinch and grip strength should be carefully considered in setting goals for conservative or surgical treatment of the rheumatoid hand. In addition, reported stiffness of the hands, malalignment of the MCP joints, and flexion and deformity of the thumb were the most consistent indicators of impaired hand function.