Equine induced pluripotent stem cells are responsive to inflammatory cytokines before and after differentiation into musculoskeletal cell types.

Persistent inflammation is associated with the poor regeneration of musculoskeletal tissues. Embryonic stem cells (ESCs) have an attenuated response to inflammatory cytokines, but there are mixed reports on the response of induced pluripotent stem cells (iPSCs) to inflammation. Horses provide a relevant large animal model for studying musculoskeletal tissue diseases and the testing of novel therapies. The aim of this study was to determine if equine iPSCs are responsive to the inflammatory cytokines IL-1ß, TNFα and IFN-γ in their undifferentiated state, or following differentiation into tendon and cartilage-like cells. We demonstrated that in undifferentiated iPSCs, the cytokines induce NF-κB P65 and STAT1 nuclear translocation which leads to cell death, decreased OCT4 expression and increased expression of inflammatory genes. Following differentiation towards cartilage-like cells exposure to the cytokines resulted in STAT1 nuclear translocation, changes in cartilage gene expression and increased expression of matrix metalloproteinases (MMPs) and inflammatory genes. Exposure of iPSC-derived tendon-like cells to the cytokines resulted nuclear translocation of NF-κB P65 and STAT1, altered tendon gene expression, increased MMP expression and increased expression of inflammatory genes. Equine iPSCs are therefore capable of responding to inflammatory stimulation and this may have relevance for their future clinical application.

Prediction of the Renal Organic Anion Transporter 1 (OAT1)- Mediated Drug Interactions for LY404039, the Active Metabolite of Pomaglumetad Methionil.

PURPOSE: The objective of this work was to demonstrate that clinical OAT1-mediated DDIs can be predicted using physiologically based pharmacokinetic (PBPK) modeling. METHODS: LY404039 is a metabotropic glutamate receptor 2/3 agonist and the active moiety of the prodrug pomaglumetad methionil (LY2140023). After oral administration, pomaglumetad methionil is rapidly taken up by enterocytes via PEPT1 and once absorbed, converted to LY404039 via membrane dehydropeptidase 1 (DPEP1). LY404039 is renally excreted by both glomerular filtration and active secretion and in vitro studies showed that the active secretion of LY404039 was mediated by the organic anion transporter 1 (OAT1). Both clinical and in vitro data were used to build a PBPK model to predict OAT1-mediated DDIs. RESULTS: In vitro inhibitory potencies (IC50) of the known OAT inhibitors, probenecid and ibuprofen, were determined to be 4.00 and 2.63 µM, respectively. Subsequently, clinical drug-drug interaction (DDI) study showed probenecid reduced the renal clearance of LY404039 by 30 to 40%. The PBPK bottom-up model, predicted a renal clearance that was approximately 20% lower than the observed one. The middle-out model, using an OAT1 relative activity factor (RAF) of 3, accurately reproduced the renal clearance of LY404039 and pharmacokinetic (PK) changes of LY404039 in the presence of probenecid. CONCLUSIONS: OAT1- mediated DDIs can be predicted using in vitro measured IC50 and PBPK modeling. The effect of ibuprofen was predicted to be minimal (AUC ratio of 1.15) and not clinically relevant.

Abemaciclib Inhibits Renal Tubular Secretion Without Changing Glomerular Filtration Rate.

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6, is indicated for metastatic breast cancer treatment. Reversible increases in serum creatinine levels of ~15-40% over baseline have been observed following abemaciclib dosing. This study assessed the in vitro and clinical inhibition of renal transporters by abemaciclib and its metabolites using metformin (a clinically relevant transporter substrate), in a clinical study that quantified glomerular filtration and iohexol clearance. In vitro, abemaciclib inhibited metformin uptake by organic cation transporter 2, multidrug and toxin extrusion (MATE)1, and MATE2-K transporters with a half-maximal inhibitory concentration of 0.4-3.8 µM. Clinically, abemaciclib significantly increased metformin exposure but did not significantly affect measured glomerular filtration rate, serum neutrophil gelatinase-associated lipocalin (NGAL), serum cystatin-C, or the urinary markers of kidney tubular injury, NGAL and kidney injury molecule-1.

Can Bile Salt Export Pump Inhibition Testing in Drug Discovery and Development Reduce Liver Injury Risk? An International Transporter Consortium Perspective.

Bile salt export pump (BSEP) inhibition has emerged as an important mechanism that may contribute to the initiation of human drug-induced liver injury (DILI). Proactive evaluation and understanding of BSEP inhibition is recommended in drug discovery and development to aid internal decision making on DILI risk. BSEP inhibition can be quantified using in vitro assays. When interpreting assay data, it is important to consider in vivo drug exposure. Currently, this can be undertaken most effectively by consideration of total plasma steady state drug concentrations (Css,plasma ). However, because total drug concentrations are not predictive of pharmacological effect, the relationship between total exposure and BSEP inhibition is not causal. Various follow-up studies can aid interpretation of in vitro BSEP inhibition data and may be undertaken on a case-by-case basis. BSEP inhibition is one of several mechanisms by which drugs may cause DILI, therefore, it should be considered alongside other mechanisms when evaluating possible DILI risk.

Prediction of Transporter-Mediated Drug-Drug Interactions for Baricitinib.

Baricitinib, an oral selective Janus kinase 1 and 2 inhibitor, undergoes active renal tubular secretion. Baricitinib was not predicted to inhibit hepatic and renal uptake and efflux drug transporters, based on the ratio of the unbound maximum eliminating-organ inlet concentration and the in vitro half-maximal inhibitory concentrations (IC50 ). In vitro, baricitinib was a substrate for organic anion transporter (OAT)3, multidrug and toxin extrusion protein (MATE)2-K, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). Probenecid, a strong OAT3 inhibitor, increased the area under the concentration-time curve from time zero to infinity (AUC[0-∞] ) of baricitinib by twofold and decreased renal clearance to 69% of control in healthy subjects. Physiologically based pharmacokinetic (PBPK) modeling reproduced the renal clearance of baricitinib and the inhibitory effect of probenecid using the in vitro IC50 value of 4.4 µM. Using ibuprofen and diclofenac in vitro IC50 values of 4.4 and 3.8 µM toward OAT3, 1.2 and 1.0 AUC(0-∞) ratios of baricitinib were predicted. These predictions suggest clinically relevant drug-drug interactions (DDIs) with ibuprofen and diclofenac are unlikely.

In Vitro and Clinical Evaluations of the Drug-Drug Interaction Potential of a Metabotropic Glutamate 2/3 Receptor Agonist Prodrug with Intestinal Peptide Transporter 1.

Despite peptide transporter 1 (PEPT1) being responsible for the bioavailability for a variety of drugs, there has been little study of its potential involvement in drug-drug interactions. Pomaglumetad methionil, a metabotropic glutamate 2/3 receptor agonist prodrug, utilizes PEPT1 to enhance absorption and bioavailability. In vitro studies were conducted to guide the decision to conduct a clinical drug interaction study and to inform the clinical study design. In vitro investigations determined the prodrug (LY2140023 monohydrate) is a substrate of PEPT1 with Km value of approximately 30 µM, whereas the active moiety (LY404039) is not a PEPT1 substrate. In addition, among the eight known PEPT1 substrates evaluated in vitro, valacyclovir was the most potent inhibitor (IC50 = 0.46 mM) of PEPT1-mediated uptake of the prodrug. Therefore, a clinical drug interaction study was conducted to evaluate the potential interaction between the prodrug and valacyclovir in healthy subjects. No effect of coadministration was observed on the pharmacokinetics of the prodrug, valacyclovir, or either of their active moieties. Although in vitro studies showed potential for the prodrug and valacyclovir interaction via PEPT1, an in vivo study showed no interaction between these two drugs. PEPT1 does not appear to easily saturate because of its high capacity and expression in the intestine. Thus, a clinical interaction at PEPT1 is unlikely even with a compound with high affinity for the transporter.

Predicting bifurcation angle effect on blood flow in the microvasculature.

Since blood viscosity is a basic parameter for understanding hemodynamics in human physiology, great amount of research has been done in order to accurately predict this highly non-Newtonian flow property. However, previous works lacked in consideration of hemodynamic changes induced by heterogeneous vessel networks. In this paper, the effect of bifurcation on hemodynamics in a microvasculature is quantitatively predicted. The flow resistance in a single bifurcation microvessel was calculated by combining a new simple mathematical model with 3-dimensional flow simulation for varying bifurcation angles under physiological flow conditions. Interestingly, the results indicate that flow resistance induced by vessel bifurcation holds a constant value of approximately 0.44 over the whole single bifurcation model below diameter of 60µm regardless of geometric parameters including bifurcation angle. Flow solutions computed from this new model showed substantial decrement in flow velocity relative to other mathematical models, which do not include vessel bifurcation effects, while pressure remained the same. Furthermore, when applying the bifurcation angle effect to the entire microvascular network, the simulation results gave better agreements with recent in vivo experimental measurements. This finding suggests a new paradigm in microvascular blood flow properties, that vessel bifurcation itself, regardless of its angle, holds considerable influence on blood viscosity, and this phenomenon will help to develop new predictive tools in microvascular research.

Prediction of renal transporter mediated drug-drug interactions for pemetrexed using physiologically based pharmacokinetic modeling.

Pemetrexed, an anionic anticancer drug with a narrow therapeutic index, is eliminated mainly by active renal tubular secretion. The in vitro to in vivo extrapolation approach used in this work was developed to predict possible drug-drug interactions (DDIs) that may occur after coadministration of pemetrexed and nonsteroidal anti-inflammatory drugs (NSAIDs), and it included in vitro assays, risk assessment models, and physiologically based pharmacokinetic (PBPK) models. The pemetrexed transport and its inhibition parameters by several NSAIDs were quantified using HEK-PEAK cells expressing organic anion transporter (OAT) 3 or OAT4. The NSAIDs were ranked according to their DDI index, calculated as the ratio of their maximum unbound concentration in plasma over the concentration inhibiting 50% (IC50) of active pemetrexed transport. A PBPK model for ibuprofen, the NSAID with the highest DDI index, was built incorporating active renal secretion in Simcyp Simulator. The bottom-up model for pemetrexed underpredicted the clearance by 2-fold. The model we built using a scaling factor of 5.3 for the maximal uptake rate (Vmax) of OAT3, which estimated using plasma concentration profiles from patients given a 10-minute infusion of 500 mg/m(2) of pemetrexed supplemented with folic acid and vitamin B12, recovered the clinical data adequately. The observed/predicted increases in Cmax and the area under the plasma-concentration time curve (AUC0-inf) of pemetrexed when ibuprofen was coadministered were 1.1 and 1.0, respectively. The coadministration of all other NSAIDs was predicted to have no significant impact on the AUC0-inf based on their DDI indexes. The PBPK model reasonably reproduced pemetrexed concentration time profiles in cancer patients and its interaction with ibuprofen.

Polarization characteristics of semipolar (112̄2) InGaN/GaN quantum well structures grown on relaxed InGaN buffer layers and comparison with experiment.

Partial strain relaxation effects on polarization ratio of semipolar (112̄2) InxGa1−xN/GaN quantum well (QW) structures grown on relaxed InGaN buffers were investigated using the multiband effective-mass theory. The absolute value of the polarization ratio gradually decreases with increasing In composition in InGaN buffer layer when the strain relaxation ratio (ε0y'y'−εy'y')/ε0y'y' along y'-axis is assumed to be linearly proportional to the difference of lattice constants between the well and the buffer layer. Also, it changes its sign for the QW structure grown on InGaN buffer layer with a relatively larger In composition (x > 0.07). These results are in good agreement with the experiment. This can be explained by the fact that, with increasing In composition in the InGaN subsrate, the spontaneous emission rate for the y'-polarization gradually increases while that for x'-polarization decreases due to the decrease in a matrix element at the band-edge (k‖ = 0).

A solid state nanopore device for investigating the magnetic properties of magnetic nanoparticles.

In this study, we explored magnetic nanoparticles translocating through a nanopore in the presence of an inhomogeneous magnetic field. By detecting the ionic current blockade signals with a silicon nitride nanopore, we found that the translocation velocity that is driven by magnetic and hydrodynamic forces on a single magnetic nanoparticle can be accurately determined and is linearly proportional to the magnetization of the magnetic nanoparticle. Thus, we obtained the magneto-susceptibility of an individual nanoparticle and the average susceptibility over one hundred particles within a few minutes.

Variability in P-glycoprotein inhibitory potency (IC50) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Genotyping of plant and animal samples without prior DNA purification.

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS) is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

3-D simulation of nanopore structure for DNA sequencing.

In this paper, we propose a method for simulating nanopore structure by using conventional 3-D simulation tool to mimic the I-V behavior of the nanopore structure. In the simulation, we use lightly doped silicon for ionic solution where some parameters like electron affinity and dielectric constant are fitted to consider the ionic solution. By using this method, we can simulate the I-V behavior of nanopore structure depending on the location and the size of the sphere shaped silicon oxide which is considered to be an indicator of a DNA base. In addition, we simulate an Ionic Field Effect Transistor (IFET) which has basically the nanopore structure, and show that the simulated curves follow sufficiently the I-V behavior of the measurement data. Therefore, we think it is reasonable to apply parameter modeling mentioned above to simulate nanopore structure. The key idea is to modify electron affinity of silicon which is used to mimic the KCl solution to avoid band bending and depletion inside the nanopore. We could efficiently utilize conventional 3-D simulation tool to simulate the I-V behavior of nanopore structures.

Revelation of the molecular assembly of the nanoporous metal organic framework ZIF-8.

Crystalline nanoporous materials are one of the most important families of complex functional material. Many questions pertaining to the molecular assembly mechanism of the framework of these materials remain unanswered. Only recently has it become possible to answer definitively some of these questions by observation of growing nanoscopic surface features on metal organic frameworks (MOFs) through use of in situ atomic force microscopy (AFM). Here we reveal that a growth process of a MOF, zeolitic imidazolate framework ZIF-8, occurs through the nucleation and spreading of successive metastable unenclosed substeps to eventually form stable surface steps of the enclosed framework structure and that this process is reliant on the presence of nonframework species to bridge the developing pores during growth. The experiments also enable identification of some of the fundamental units in the growth process and the stable crystal surface plane. The former findings will be applicable to numerous nanoporous materials and support efforts to synthesize and design new frameworks and to control the crystal properties of these materials.

Unresectable huge sternal and mediastinal metastasis of follicular thyroid carcinoma; radiotherapy as first-line and palliative therapy.

Distant metastases as initial manifestation of follicular thyroid carcinoma is rare. We report a case of an unusual initial presentation of follicular thyroid carcinoma on follow-up. A 52- year-old woman presented with a 12-month history of progressively enlarging mass in the anterior chest wall. The mass was fixed to the chest wall, measuring 12 x 10 cm in diameter. Computed tomography demonstrated a lobulated soft-tissue mass (17 x 11 x 6 cm) destructing sternum and extending into the anterior mediastinum. There was no lung metastasis. Invasion of tumor to the ascending aorta, superior vena cava, and right atrium could not be excluded. Multiple lymph nodes were observed in the supraclavicular regions. Ultrasonography of the thyroid gland showed 46 x 37 mm nodule in the left lobe with milimetric gross calcifications and cystic-necrotic areas. Hyperthyroidism was detected. Biopsy from this nodule and the sternal mass revealed typical histology of follicular carcinoma. She was considered inoperable. Since there was huge tumor burden and iodinated contrast exposure for several times during evaluation, we decided to treat the patient with external beam radiotherapy (EBRT) rather than radioiodine as first-line therapy. After a course of conventional radiation with 50 Gy in 25 fractions over 4 weeks, encompassing the thyroid bed and the gross disease, tumor regressed remarkably in 6 months. In conclusion, when surgical resection is not possible, EBRT may be used for palliative purpose to obtain local control for extensive disease as first-line therapy. The indications of EBRT for differentiated thyroid cancer still remain poorly defined.

Factors associated with actual long-term survival following soft tissue sarcoma pulmonary metastasectomy.

AIMS: To identify clinicopathologic and treatment variables associated with long-term overall survival (OS) in soft tissue sarcoma (STS) patients with lung metastases undergoing pulmonary metastasectomy (PM). METHODS: Retrospective review of 94 STS PM patients with an actual follow-up > or = 5 years. Data were collected on demographics, tumor features, treatment, and outcome. RESULTS: Most primary tumors were intermediate/high grade and the common histopathologies were evenly distributed. Half of the primary tumors were located on the extremities. The mean disease-free interval (DFI) from time of original diagnosis until metastases was 25 months (median 15 months). Eighteen patients had synchronous metastatic disease. Bilateral pulmonary metastases and >1 metastasis were common. The median number of metastases resected was 2.5. Thirty-four patients had extrapulmonary tumor at the time of PM; all extrapulmonary disease was resected. Negative margin resection (R0) PM was performed in 74 patients. Actual 5-year disease-free survival (DFS) and OS for all patients were 5% and 15%, respectively. For the R0 group, actual 5-year DFS and OS were 7% and 18%, respectively. R0 resection and a prolonged DFI were associated with improved OS. Patient characteristics, tumor features, local recurrence, and adjuvant therapy did not affect OS. CONCLUSIONS: Less than 20% of STS PM patients will survive 5 years. Complete resection and DFI are the most predictive factors for prolonged survival.

Epithelial-mesenchymal transition (EMT) and activated extracellular signal-regulated kinase (p-Erk) in surgically resected pancreatic cancer.

BACKGROUND: EMT or transformation to the mesenchymal phenotype plays an important role in tumor invasion and metastasis. In vitro data suggest that mesenchymal transformation may correlate with the activation of PI3 kinase and Ras/Erk pathways. We investigated the expression of EMT markers (low E-cadherin, high fibronectin, and vimentin) and their association with p-Erk in resected pancreatic cancer. METHODS: Clinical data/surgical specimens from 34 consecutive pancreatic cancer patients (pts) who underwent pancreatectomy were included. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissues using monoclonal antibodies against vimentin, fibronectin, E-cadherin, and p-Erk. The results were correlated with clinicopathological parameters and survival. Survival analysis (log-rank test, Cox proportional hazard model), categorical data analysis (Pearson's chi-square, Fisher's exact test) and Kendall's tau were performed at a significance level of 0.05. RESULTS: The patient population was formed from 13 males and 21 females, with a median age of 66 years (range 38-84 years); American Joint Committee on Cancer (AJCC) stage 1 (n = 2), 2 (n = 27), 3 (n = 5); histological grade 1 (n = 4), 2 (n = 13), 3 (n = 16), 4 (n = 1). Median survival was 15 months (95% CI: 11-24 months). Fibronectin overexpression correlated with the presence of vimentin (p = 0.0048) and activated Erk (p = 0.0264). There was a borderline association of fibronectin with worsening grade (p = 0.06). A negative association between vimentin and E-cadherin was noted (p = 0.0024). Increased fibronectin or vimentin and decreased E-cadherin correlated with poor survival. CONCLUSION: EMT is associated with poor survival in surgically resected pancreatic adenocarcinoma. A correlation between activated Erk and fibronectin was identified that may open avenues for targeted therapy for this subgroup.

Safety and efficacy of transdermal fentanyl in patients with cancer pain: phase IV, Turkish oncology group trial.

We have performed a prospective evaluation of the efficacy, safety and convenience of the transdermal therapeutic system - fentanyl (TTS-F) in Turkish cancer patients when it was newly available in Turkey. Ninety-nine patients with historically confirmed malignancy and pain entered the study; the mean age was 55.1 (16-58) years. The study duration was 28 days. Transdermal therapeutic system - fentanyl was used in opioid-naïve or pre-treated patients. Most patients reported a decrease in pain severity. Use of rescue medication decreased from day 4 to day 28. The majority of patients rated patch convenience of use as excellent. A total of 22.2% of patients experienced adverse events that were either probably related or very likely to be related to the study drug. The majority of the adverse events mentioned were related to the digestive system. Eighteen serious adverse events were reported by 13 patients. Six events were doubtfully related, and 12 events were not related to the study drug. Four patients died during the trial. None of these deaths was attributed to the study drug. In conclusion, the trial showed that TTS-F is easily managed, effective and will help to enable the appropriate opioid administration to patients who are suffering from cancer pain in Turkey.

Clinical evaluation of micro-scale chip-based PCR system for rapid detection of hepatitis B virus.

The polymerase chain reaction (PCR) is widely used to amplify a small amount of DNA in samples for genetic analysis. Rapid and accurate amplification is prerequisite for broad applications including molecular diagnostics of diseases, food safety, and biological warfare tests. We have developed a rapid real-time micro-scale chip-based PCR system, which consists of six individual thermal cycling modules capable of independent control of PCR protocols. The PCR volume is 1 microl and it takes less than 20 min to complete 40 thermal cycles. To test utility of a chip-based PCR system as a molecular diagnostic device, we have conducted the first large-scale clinical evaluation study. Three independent clinical evaluation studies (n = 563) for screening the hepatitis B virus (HBV) infection, the most popular social epidemic disease in Asia, showed an excellent sensitivity, e.g. 94%, and specificity, e.g. 93%, demonstrating micro-scale chip-based PCR can be applied in molecular diagnostics.

Characterization of DNA immobilization and subsequent hybridization using in situ quartz crystal microbalance, fluorescence spectroscopy, and surface plasmon resonance.

We have characterized the immobilization of thiol-modified oligomers on Au surfaces and subsequent hybridization with a perfectly matched or single-base mismatched target using a quartz crystal microbalance (QCM) and fluorescence spectroscopy. The surface density of immobilized probe molecules and the hybridization efficiency depending on the type of buffer and salt concentration were investigated. We observed some ambiguities in surface coverage deduced from QCM measurement and adopted a complementary fluorescence displacement method. Direct comparison of surface coverage deduced from frequency change in QCM measurement and determined by the fluorescence exchange reaction revealed that QCM results are highly overestimated and the amount of overestimation strongly depends on the type of buffer and the structure of the film. Discrimination capability of the surface attached 15-mer probe was also examined using a single-base mismatched target at various hybridization temperatures. Hybridization efficiency depending on the type of single base mismatch was investigated using surface plasmon resonance (SPR).