The Role of Nonmetastatic Lymph Nodes in the Survival of Colorectal Cancer: A Systematic Review.

Objective: In this review, we aim to provide an overview of literature on lymph node (LN) histomorphological features and their relationship with the prognosis in colorectal cancer (CRC). Background: Lymph nodes play a crucial role in the treatment and prognosis of CRC. The presence of LN metastases considerably worsens the prognosis in CRC patients. Literature has shown that the total number of LNs and the number negative LNs (LNnegs) has prognostic value in CRC patients. In esophageal carcinoma, LN size seems to be surrogate of the host antitumor response and a potentially clinically useful new prognostic biomarker for (y)pN0 esophageal carcinoma. Methods: A comprehensive search was performed in Pubmed, Embase, Medline, CINAHL, and the Cochrane library in March 2021. The PRISMA guidelines were followed. Only studies focusing on histomorphological features and LN size and their relation to overall survival were selected. Results: A total of 9 unique articles met all inclusion criteria and were therefore included in this systematic review. Six of these studies investigated HMF (eg, paracortical hyperplasia, germinal center predominance, and sinus histiocytosis) and 4 studies LNneg size and their relationship with overall survival. The presence of paracortical hyperplasia and an increased number of large LNnegs is related to a more favorable prognosis in CRC. Conclusion: The results of this systematic review seem to support the hypothesis that there is a relationship between the host antitumor response reflected in different histomorphological reaction patterns visible in LNnegs and LNneg size related to survival in CRC patients.

Dietary and serum tyrosine, white matter microstructure and inter-individual variability in executive functions in overweight adults: Relation to sex/gender and age.

Tyrosine (tyr), the precursor of the neurotransmitter dopamine, is known to modulate cognitive functions including executive attention. Tyr supplementation is suggested to influence dopamine-modulated cognitive performance. However, results are inconclusive regarding the presence or strength and also the direction of the association between tyr and cognitive function. This pre-registered cross-sectional analysis investigates whether diet-associated serum tyr relates to executive attention performance, and whether this relationship is moderated by differences in white matter microstructure. 59 healthy, overweight, young to middle-aged adults (20 female sex/gender group, 28.3 ± 6.6 years, BMI: 27.3 ± 1.5 kg/m2) drawn from a longitudinal study reported dietary habits, donated blood and completed diffusion-weighted brain magnetic resonance imaging and the attention network test. Main analyses were performed using linear regressions and non-parametric voxel-wise inference testing. Confirmatory analyses did neither support an association between dietary and serum tyr nor a relationship between relative serum tyr/large neutral amino acids (LNAA) levels or white matter microstructure and executive attention performance. However, exploratory analyses revealed higher tyr intake, higher serum tyr and better executive attention performance in the male sex/gender group. In addition, older age was associated with higher dietary tyr intake and lower fractional anisotropy in a widespread cluster across the brain. Finally, a positive association between relative serum tyr/LNAA level and executive attention performance was found in the male sex/gender group when accounting for age effects. Our analysis advances the field of dopamine-modulated cognitive functions by revealing sex/gender and age differences which might be diet-related. Longitudinal or intervention studies and larger sample sizes are needed to provide more reliable evidence for links between tyr and executive attention.

EBMT prospective observational study on allogeneic hematopoietic stem cell transplantation in T-prolymphocytic leukemia (T-PLL).

Preliminary data suggest that allogeneic stem cell transplantation (allo-SCT) may be effective in T-prolymphocytic leukemia (T-PLL). The purpose of the present observational study was to assess the outcome of allo-SCT in patients aged 65 years or younger with a centrally confirmed diagnosis of T-PLL. Patients were consecutively registered with the EBMT at the time of transplantation and followed by routine EBMT monitoring but with an extended dataset. Between 2007 and 2012, 37 evaluable patients (median age 56 years) were accrued. Pre-treatment contained alemtuzumab in 95% of patients. Sixty-two percent were in complete remission (CR) at the time of allo-SCT. Conditioning contained total body irradiation with 6 Gy or more (TBI6) in 30% of patients. With a median follow-up of 50 months, the 4-year non-relapse mortality, relapse incidence, progression-free (PFS) and overall survival were 32, 38, 30 and 42%, respectively. By univariate analysis, TBI6 in the conditioning was the only significant predictor for a low relapse risk, and an interval between diagnosis and allo-SCT of more than 12 months was associated with a lower NRM. This study confirms for the first time prospectively that allo-SCT can provide long-term disease control in a sizable albeit limited proportion of patients with T-PLL.

Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling.

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP-/- mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, ß-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.

Pharmacological Treatment with Annexin A1 Reduces Atherosclerotic Plaque Burden in LDLR-/- Mice on Western Type Diet.

OBJECTIVE: To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR-/- mice. METHODS: Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of 99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry. RESULTS: Hr-anxA1 bound PS in Ca2+-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating 99mTc-hr-anxA1 were <10 minutes and approximately 6 hours for intravenously (IV) and intraperitoneally (IP) administered hr-anxA1, respectively. Pharmacological treatment with hr-anxA1 had no significant effect on initiation of plaque formation (-33%; P = 0.21)(Group I) but significantly attenuated progression of existing plaques of aortic arch and subclavian artery (plaque size -50%, P = 0.005; necrotic core size -76% P = 0.015, hr-anxA1 vs vehicle) (Group P). CONCLUSION: Hr-anxA1 may offer pharmacological means to treat chronic atherogenesis by reducing FPR-2 dependent neutrophil rolling and adhesion to activated endothelial cells and by reducing total plaque inflammation.

Gla-rich protein acts as a calcification inhibitor in the human cardiovascular system.

OBJECTIVE: Vascular and valvular calcifications are pathological processes regulated by resident cells, and depending on a complex interplay between calcification promoters and inhibitors, resembling skeletal metabolism. Here, we study the role of the vitamin K-dependent Gla-rich protein (GRP) in vascular and valvular calcification processes. APPROACH AND RESULTS: Immunohistochemistry and quantitative polymerase chain reaction showed that GRP expression and accumulation are upregulated with calcification simultaneously with osteocalcin and matrix Gla protein (MGP). Using conformation-specific antibodies, both γ-carboxylated GRP and undercarboxylated GRP species were found accumulated at the sites of mineral deposits, whereas undercarboxylated GRP was predominant in calcified aortic valve disease valvular interstitial cells. Mineral-bound GRP, MGP, and fetuin-A were identified by mass spectrometry. Using an ex vivo model of vascular calcification, γ-carboxylated GRP but not undercarboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation through α-smooth muscle actin upregulation and osteopontin downregulation. Immunoprecipitation assays showed that GRP is part of an MGP-fetuin-A complex at the sites of valvular calcification. Moreover, extracellular vesicles released from normal vascular smooth muscle cells are loaded with GRP, MGP, and fetuin-A, whereas under calcifying conditions, released extracellular vesicles show increased calcium loading and GRP and MGP depletion. CONCLUSIONS: GRP is an inhibitor of vascular and valvular calcification involved in calcium homeostasis. Its function might be associated with prevention of calcium-induced signaling pathways and direct mineral binding to inhibit crystal formation/maturation. Our data show that GRP is a new player in mineralization competence of extracellular vesicles possibly associated with the fetuin-A-MGP calcification inhibitory system. GRP activity was found to be dependent on its γ-carboxylation status, with potential clinical relevance.

Insights into the association of Gla-rich protein and osteoarthritis, novel splice variants and γ-carboxylation status.

SCOPE: Gla-rich protein (GRP) is a vitamin K dependent protein, characterized by a high density of γ-carboxylated Glu residues, shown to accumulate in mouse and sturgeon cartilage and at sites of skin and vascular calcification in humans. Therefore, we investigated the involvement of GRP in pathological calcification in osteoarthritis (OA). METHODS AND RESULTS: Comparative analysis of GRP patterning at transcriptional and translational levels was performed between controls and OA patients. Using a RT-PCR strategy we unveiled two novel splice variants in human-GRP-F5 and F6-potentially characterized by the loss of full γ-carboxylation and secretion functional motifs. GRP-F1 is shown to be the predominant splice variant expressed in mouse and human adult tissues, particularly in OA cartilage, while an overexpressing human cell model points it as the major γ-carboxylated isoform. Using validated conformational antibodies detecting carboxylated or undercarboxylated GRP (c/uc GRP), we have demonstrated cGRP accumulation in controls, whereas ucGRP was the predominant form in OA-affected tissues, colocalizing at sites of ectopic calcification. CONCLUSION: Overall, our results indicate the predominance of GRP-F1, and a clear association of ucGRP with OA cartilage and synovial membrane. Levels of vitamin K should be further assessed in these patients to determine its potential therapeutic use as a supplement in OA treatment.

The realm of vitamin K dependent proteins: shifting from coagulation toward calcification.

In the past few decades vitamin K has emerged from a single-function "haemostasis vitamin" to a "multi-function vitamin." The use of vitamin K antagonists (VKA) inevitably showed that the inhibition was not restricted to vitamin K dependent coagulation factors but also synthesis of functional extrahepatic vitamin K dependent proteins (VKDPs), thereby eliciting undesired side effects. Vascular calcification is one of the recently revealed detrimental effects of VKA. The discovery that VKDPs are involved in vascular calcification has propelled our mechanistic understanding of this process and has opened novel avenues for diagnosis and treatment. This review addresses mechanisms of VKDPs and their significance for physiological and pathological calcification.

Destruction and formation of PCDD/Fs in a fluidised bed combustor co-incinerating automotive shredder residue with refuse derived fuel and wastewater treatment sludge.

During an eight day trial automotive shredder residue (ASR) was added to the usual waste feed of a Fluidized Bed Combustor (FBC) for waste-to-energy conversion; the input waste mix consisted of 25% ASR, 25% refuse-derived fuel (RDF) and 50% wastewater treatment (WWT) sludge. All inputs and outputs were sampled and the concentration of the 17 PCDD/Fs with TEF-values was determined in order to obtain "PCDD/F fingerprints". The ASR contained approximately 9000 ng PCDD/Fs/kg(DW), six times more than the RDF and 10 times more than the WWT sludge. The fingerprint of ASR and RDF was dominated by HpCDD and OCDD, which accounted for 90% of the total PDDD/F content, whereas the WWT sludge contained relatively more HpCDFs and OCDF (together 70%). The flue gas cleaning residue (FGCR) and fly and boiler ash contained approximately 30,000 and 2500 ng PCDD/Fs/kg(DW), respectively. The fingerprints of these outputs were also dominated by HpCDFs and OCDF. The bottom ash contained only OCDD and OCDF, in total 8 ng PCDD/Fs/kg (DW). From the comparison of the bottom ash fingerprints with the fingerprints of the other output fractions and of the inputs, it could be concluded that the PCDD/Fs in the waste were destroyed and new PCDD/Fs were formed in the post combustion process by de novo synthesis. During the ASR-co-incineration, the PCDD/F congener concentrations in the fly and boiler ash, FGCR and flue gas were 1.25-10 times higher compared to the same output fractions generated during incineration of the usual waste mix (70% RDF and 30% WWT sludge). The concentration of the higher chlorinated PCDD/Fs increased most. As these congeners have the lowest TEF-factors, the total PCDD/F output, expressed in kg TEQ/year, of the FBC did not increase significantly when ASR was co-incinerated. Due to the relatively high copper levels in the ASR, the copper concentrations in the FBCs outputs increased. As copper catalysis the de novo syntheses, this could explain the increase in PCDD/F concentrations in these outputs.

Mass balance for POPs in a real scale fluidized bed combustor co-incinerating automotive shredder residue.

The European directive 2000/53/EC implies a "reuse and recovery" rate for end-of-life vehicles (ELVs) of 95% to be reached by the year 2015. One of the options to increase the actual average European "reuse and recovery" rate of approximately 78% (EU 15, 2008) is incineration of automotive shredder residue (ASR) with energy-recovery. The mass balance and the congener fingerprints for PCDD/Fs, dioxin-like PCBs, PCBs and PAHs in a real scale fluidized bed combustor (FBC) incinerating 25% ASR with 25% refuse derived fuel (RDF) and 50% waste water treatment sludge (WWT sludge) were investigated. The PCDD/F, dioxin-like PCB, PCB and PAH concentrations in this input waste mix were more than hundred times higher than in the usual waste feed of the incinerator (30% RFD and 70% WWT sludge). In the outputs of the FBC, however, the concentrations of these POP groups were comparable or only slightly higher than in the outputs generated during the incineration of the usual waste feed. The considered POPs in the waste were destroyed efficiently and the formation of new POPs during cooling of the flue gas appeared to a large extent independent of the POP concentrations in the incinerated waste.

Mass balance for POPs in hazardous and municipal solid waste incinerators.

The amount of different persistent organic pollutants (POPs) in the input of waste incinerators was compared to that in the output. Three cases were considered: a rotary kiln incinerating hazardous waste, a grate furnace incinerating municipal solid waste (MSW) and the same grate furnace co-incinerating plastics of waste of electrical and electronic equipment (WEEE) and automotive shredder residue (ASR) with MSW. The mass balance for PCBs in the rotary kiln indicates that these POPs are destroyed effectively during incineration. The grate furnace can be a sink or source of PCDD/Fs and PCBs depending on the concentrations in the incinerated waste. In order to compare the total amount of POPs in input and output, a methodology was developed whereby the amount of POPs was weighed according to minimal risk doses (MRDs) or cancer potency factors. For both incinerators the PCDD/Fs, PCBs and polyaromatic hydrocarbons (PAHs) are the main contributors to total weighed POP output. In MSW, the PCDD/Fs, PBDD/Fs and polybrominated diphenylethers (PBDEs) are the main contributors to the weighed POP input. The ratios of the weighed POP-input over -output clearly indicate that the rotary kiln incinerating hazardous waste is a weighed POP sink. The grate furnace incinerating MSW is a weighed POP sink or source depending on the POP-concentrations in the waste, but the difference between output and input is rather limited. When e.g. ASR and plastics of WEEE, containing high concentrations of PBDEs and PCBs, are co-incinerated in the grate furnace, it is clearly a weighed POP sink.

Quantification of ventilation characteristics of a helmet.

Despite the augmented safety offered by wearing a cyclist crash helmet, many cyclists still refuse to wear one because of the thermal discomfort that comes along with wearing it. In this paper, a method is described that quantifies the ventilation characteristics of a helmet using tracer gas experiments. A Data-Based Mechanistic model was applied to provide a physically meaningful description of the dominant internal dynamics of mass transfer in the imperfectly mixed fluid under the helmet. By using a physical mass balance, the local ventilation efficiency could be described by using a single input-single output system. Using this approach, ventilation efficiency ranging from 0.06 volume refreshments per second (s(-1)) at the side of the helmet to 0.22s(-1) at the rear ventilation opening were found on the investigated helmet. The zones at the side were poorly ventilated. The influence of the angle of inclination on ventilation efficiency was dependent on the position between head and helmet. General comfort of the helmet can be improved by increasing the ventilation efficiency of fresh air at the problem zones.

Comparison of thermal comfort performance of two different types of road vehicle climate control systems.

The performance of climate control systems in vehicles becomes more and more important, especially against the background of the important relationship between compartment climate and driver mental condition and, thus, traffic safety. The performance of two different types of climate control systems, an un-air-conditioned heating/cooling device (VW) and an air-conditioning climate control unit (BMW), is compared using modern and practical evaluation techniques quantifying both the dynamic 3-D temperature distribution and the local air refreshment rate. Both systems suffer from considerable temperature gradients: temperature gradients in the U-AC (VW) car up to 8-9 degrees C are encountered, while the AC (BMW) delivers clear improvement resulting in temperature gradients of 5-6 degrees C. The experiments clearly demonstrate the effect of the presence of even a single passenger on the thermal regime, increasing the existing thermal discrepancies in the compartment with 15% independent of ventilation rate. Furthermore, in terms of air refreshment rates in the vehicle compartment, an air-conditioning unit halves the air refreshment time at all positions in the vehicle cabin, delivering a significant improvement in terms of human comfort. Similarly, extra air inlets in the back compartment of a car deliver progress in terms of cabin refreshment rate (93 s down to 50 s).

[Generalized lymphadenitis associated with Hashimoto's thyroiditis]. / Generalisierte Lymphadenitis mit Hashimoto-Thyreoiditis.

HISTORY AND ADMISSION FINDINGS: A 26-year-old Pakistani woman was admitted to hospital with fever, nausea and vomiting, abdominal pain and general weakness for the previous two weeks, but no diarrhea or constipation. Antibiotic therapy as an out-patient had not provided relief. She also had enlarged cervical lymph nodes. She reported that she had always been in good health before onset of the described symptoms. INVESTIGATIONS: Laboratory tests revealed pancytopenia and a decrease in C3 and C4. The ANA titer was positive. The serum was positive for preceding infections with EBV, CMV, Chlamydia trachomatis, HHV type 6, and toxoplasmosis. There was no evidence of current or previous infections with HIV, Bartonella henselae, Treponema pallidum, Listeria, Hantavirus, Leishmania or dengue fever. The patient's symptoms were found to be associated with Hashimoto's thyroiditis in conjunction with latent hypothyroidism. Abdominal, thoracic and cervical CT scan revealed enlarged lymph nodes, up to 2 cm in size, and enlarged spleen and liver. THERAPY AND CLINICAL COURSE: Histopathology of a cervical lymph node showed features of Kikuchi (Kikuchi-Fujimoto) disease (histiocytic necrotizing lymphadenitis). Prednisone administration (2 mg/kg body weight) led to the patient's rapid recovery. CONCLUSION: Kikuchi disease should be considered in patients with cervical but also general lymphadenopathy, after exclusion of other causes. The specific diagnosis is based on biopsy of affected lymph nodes.

Quantification of the heat exchange of chicken eggs.

In the incubation process of domestic avian eggs, the development of the embryo is mainly influenced by the physical microenvironment around the egg. Only small spatiotemporal deviations in the optimal incubator air temperature are allowed to optimize hatchability and hatchling quality. The temperature of the embryo depends on 3 factors: (1) the air temperature, (2) the exchange of heat between the egg and its microenvironment and (3) the time-variable heat production of the embryo. Theoretical estimates on the heat exchange between an egg and its physical microenvironment are approximated using equations that assume an approximate spherical shape for eggs. The objective of this research was to determine the heat transfer between the eggshell and its microenvironment and then compare this value to various theoretical estimates. By using experimental data, the overall and the convective heat transfer coefficients were determined as a function of heat production, air humidity, air speed, and air temperature. Heat transfer was not affected by air humidity but solely by air temperature, embryonic heat generation, and air speed and flow around eggs. Also, heat transfer in forced-air incubators occurs mainly by convective heat loss, which is dependent on the speed of airflow. A vertical airflow is more efficient than a horizontal airflow in transferring heat from the egg. We showed that describing an egg as a sphere underestimated convective heat transfer by 33% and was, therefore, too simplistic to accurately assess actual heat transfer from real eggs.

Modeling the internal dynamics of energy and mass transfer in an imperfectly mixed ventilated airspace.

UNLABELLED: The present paper outlines a modeling approach, which has been developed to model the internal dynamics of heat and moisture transfer in an imperfectly mixed ventilated airspace. The modeling approach, which combines the classical heat and moisture balance differential equations with the use of experimental time-series data, provides a physically meaningful description of the process and is very useful for model-based control purposes. The paper illustrates how the modeling approach has been applied to a ventilated laboratory test room with internal heat and moisture production. The results are evaluated and some valuable suggestions for future research are forwarded. PRACTICAL IMPLICATIONS: The modeling approach outlined in this study provides an ideal form for advanced model-based control system design. The relatively low number of parameters makes it well suited for model-based control purposes, as a limited number of identification experiments is sufficient to determine these parameters. The model concept provides information about the air quality and airflow pattern in an arbitrary building. By using this model as a simulation tool, the indoor air quality and airflow pattern can be optimized.

Quantification and control of the spatiotemporal gradients of air speed and air temperature in an incubator.

Around the optimal incubator air temperature only small spatiotemporal deviations are allowed. However, air speed and air temperature are not uniformly distributed in the total volume of the incubator due to obstruction of the eggs and egg trays. The objectives of this research were (1) to quantify the spatiotemporal gradients in temperature and velocity and (2) to develop and validate a control algorithm to increase the uniformity in temperature during the entire incubation process. To improve the uniformity of air temperature, the airflow pattern and the air quality need to be controlled more optimally. These data show that the air temperature between the eggs at a certain position in a large incubator is the result of (1) the mean air temperature of the incubator; (2) the exchange of heat between the egg and its micro-environment, which is affected by the air speed at that certain position; (3) the time-variable heat production of the embryo; and (4) the heat influx or efflux as a result from the movement of hot or cold air in the incubator toward that position, which is affected by the airflow pattern. This implies that the airflow pattern needs to be controlled in a more optimal way. To maximize the uniformity of air temperature, an active and adaptive control of the three-dimensional (3-D) airflow pattern has been developed and tested. It was found to improve the spatiotemporal temperature distribution. The chance of having a temperature reading in the interval from 37.5 to 38.1 degrees C increased by 3% compared to normal operating conditions.

Miniaturization of a homogeneous fluorescence immunoassay based on energy transfer using nanotiter plates as high-density sample carriers.

The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 x 2 cm2 containing 25 x 25 wells, corresponding to approximately 15,000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelength-excitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 microg/L, or 1.11 x 10(-16) mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 microg/L, or 1.87 x 10(-13) mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 microg/L.

Covalent strategy for immobilization of DNA-microspots suitable for microarrays with label-free and time-resolved optical detection of hybridization.

Sequence-specific detection and quantification of nucleic acids are central steps in many molecular biology procedures which have also been transferred to chip-based procedures. Hybridization-based assays can be used to quantify and discriminate between DNA target sequences down to the level of single base mismatches. Arrays of DNA probes immobilized on a support enable simultaneous testing of multiple sequences of a single sample. DNA arrays can be produced either by in-situ synthesis of oligonucleotides or by immobilization of pre-assembled DNA probes. Covalent and directed immobilization improves the reproducibility and stability of DNA arrays. This is especially interesting with repeated use of transducers or chips. Procedures are described for effective covalent immobilization of pre-assembled amino-linked oligonucleotides, by use of ink-jet techniques, on a modified and heated glass surface, with addressable surface areas ranging from 0.01 mm2 to a few mm2. Almost immediate evaporation of the spotted droplets on the heated surfaces leads to very high surface hybridization capacities. The surfaces are suitable for use with a label-free detection method - reflectometric interference spectroscopy (RIfS). It is shown that hybridization capacity and non-specific interaction at these DNA-surfaces can be characterized by use of RIfS. With a consumption of less than 80 ng mm(-2) oligonucleotide and a specific hybridization capacity of more than 300 fmol mm(-2), the activated aminodextran procedure was usually suitable for setting up a DNA array with label-free detection. Non-specific interactions with random oligomers or protein (ovalbumin) were low. Up to 150 repeated regenerations (stripping) of the surfaces by acid treatment and denaturing agents, and 50 days of storage, have been possible without significant loss of hybridization capacity.