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We report the efficient single-step separation of individual platelets from unprocessed whole blood, enabling digital quantification of platelet function using interfacial platelet cytometry (iPC) on a chip. iPC is accomplished by the precision micropatterning of platelet-specific protein surfaces on solid substrates. By separating platelets from whole blood using specific binding to protein spots of a defined size, iPC implements a simple incubate-and-rinse approach, without sample preparation, that enables (1) the study of platelets in the physiological situation of interaction with a protein surface, (2) the choice of the number of platelets bound on each protein spot, from one to many, (3) control of the platelet-platelet distance, including the possibility to study noninteracting single platelets, (4) digital quantification (counting) of platelet adhesion to selected protein matrices, enabling statistical characterization of platelet subpopulations from meaningfully large numbers of single platelets, (5) the study of platelet receptor expression and spatial distribution, and (6) a detailed study of the morphology of isolated single platelets at activation levels that can be manipulated. To date, we have demonstrated 1-4 of the above list. Platelets were separated from whole blood using iPC with fibrinogen, von Willebrand factor (VWF), and anti-CD42b antibody printed "spots" ranging from a fraction of one to several platelet diameters (2-24 μm). The number of platelets captured per spot depends strongly on the protein matrix and the surface area of the spot, together with the platelet volume, morphology, and activation state. Blood samples from healthy donors, a May-Hegglin-anomaly patient, and a Glanzmann's Thrombasthenia patient were analyzed via iPC to confirm the specificity of the interaction between protein matrices and platelets. For example, the results indicate that platelets interact with fibrinogen spots only through the fibrinogen receptor (αIIbβ3) and, relevant to diagnostic applications, platelet adhesion correlates strongly with normal versus abnormal platelet function. A critical function of platelets is to adhere to regions of damage on blood vessel walls; in contrast to conventional flow cytometry, where platelets are suspended in solution, iPC enables physiologically relevant platelet bioassays based on platelet/protein-matrix interactions on surfaces. This technology should be inexpensive to implement in clinical assay format, is readily integrable into fluidic microdevices, and paves the way for high-throughput platelet assays from microliter volumes of whole blood.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are major causes of liver disease in Taiwan and have a great impact on the health of this country. This study investigated the seroprevalence of HBV and HCV in southern Taiwan. Screening programs were performed from September 1999 to August 2005 for community-based surveillance of liver disease. A total of 28,797 adults from southern Taiwan, including Kaohsiung City (n = 14,036), Kaohsiung County (n = 7,713), and Pingtung County (n = 7,048) were participated. The mean age was 50.3 ± 14.6 years (range, 20-97 years), with 41.0% were men. Hepatitis B surface antigen (HBsAg), antibody to HCV (anti-HCV), and liver function tests were performed. Among the 28,797 adults, the prevalence of HBsAg(+) was 15.1% and that for anti-HCV(+) was 8.6%. The seroprevalence of HBsAg in Kaohsiung County was 18.2%, which was higher than in Kaohsiung City (14.7%, p < 0.001) or Pingtung County (12.5%, p < 0.001). The seroprevalence of anti-HCV in Kaohsiung County was 17.2%, which was higher than in the other regions (Kaohsiung City = 5.8%, p < 0.001; Pingtung County = 4.6%, p < 0.001). The prevalence of dual HBsAg and anti-HCV was 1.1% (323 patients). Tzukuan Township in Kaohsiung County was endemic for HBsAg (19.1%, 1,026/5,375 patients), anti-HCV (22.4%, 1,203/5,375 patients), and dual HBsAg/anti-HCV (3.6%, 191/5,375 patients). Subjects with anti-HCV(+) were older and had higher alanine transaminase levels than their HBsAg(+) counterparts (p < 0.001 and p < 0.001, respectively). The current study shows the epidemiological characteristics of HBV and HCV infections among adults in southern Taiwan. Viral hepatitis infections remain widely endemic in this region., (Copyright © 2010 Elsevier. Published by Elsevier B.V. All rights reserved.)

Free electrons in a noble metal nanoparticle can be resonantly excited, leading to their collective oscillation termed as a surface plasmon. These surface plasmons enable nanoparticles to absorb light, generate heat, transfer energy, and re-radiate incident photons. Creative designs of nanoplasmonic optical antennae (i.e. plasmon resonant nanoparticles) have become a new foundation of quantitative biology and nanomedicine. This review focuses on the recent developments in dual-functional nanoplasmonic optical antennae for label-free biosensors and nanoplasmonic gene switches. Nanoplasmonic optical antennae, functioning as biosensors to significantly enhance biochemical-specific spectral information via plasmon resonance energy transfer (PRET) and surface-enhanced Raman spectroscopy (SERS), are discussed. Nanoplasmonic optical antennae, functioning as nanoplasmonic gene switches to enable spatiotemporal regulation of genetic activity, are also reviewed. Nanoplasmonic molecular rulers and integrated photoacoustic-photothermal contrast agents are also described., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A device for continuous differential impedance analysis of single cells held by a hydrodynamic cell trapping is presented. Measurements are accomplished by recording the current from two closely-situated electrode pairs, one empty (reference) and one containing a cell. We demonstrate time-dependent measurement of single cell impedance produced in response to dynamic chemical perturbations. First, the system is used to assay the response of HeLa cells to the effects of the surfactant Tween, which reduces the impedance of the trapped cells in a concentration dependent way and is interpreted as gradual lysis of the cell membrane. Second, the effects of the bacterial pore-forming toxin, Streptolysin-O are measured: a transient exponential decay in the impedance is recorded as the cell membrane becomes increasingly permeable. The decay time constant is inversely proportional to toxin concentration (482, 150, and 30 s for 0.1, 1, and 10 kU/ml, respectively). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10404-009-0534-2) contains supplementary material, which is available to authorized users.

To harness the properties of both PDMS and silica, we have demonstrated hybrid integrated PDMS microfluidic systems with fused silica capillaries. The hybrid integrated PDMS microfluidics and silica capillary (iPSC) modules exhibit a novel architecture and method for leakage free CE sample injection merely requiring a single high voltage source and one pair of electrodes. The use of the iPSC device is based on a modular approach which allows the capillary to be reused extensively whilst replacing the attached fluidic module for different experiments. Integrating fused silica capillaries with PDMS microfluidic modules allows the direct application of a wide variety of well established conventional CE protocols for separations of complex analytes. Furthermore it bears the potential for facile coupling to standard electro-spray ionization mass spectrometry (ESI-MS), letting users focus on the sample analysis rather than the development of new separation protocols. The fabrication of the iPSC module consists of a simple and quick three-step method that submerges a fused silica capillary in PDMS prepolymer. After cross linking the prepolymer and punching the inlets, the iPSC module layer can be mounted onto a microfluidic device for CE separation.

Objectives: To (1) determine the demographics of Chinese children admitted with primary spontaneous pneumothorax, (2) suggest how they may be quantified radiologically, (3) compare the difference in outcomes after their primary management by thoracentesis and chest tube insertion, and (4) review the local experience with surgical intervention for such children., Design: Retrospective, descriptive study., Setting: Acute tertiary public hospital, Hong Kong., Patients: Consecutive patients younger than 18 years and admitted with primary spontaneous pneumothorax between 1 January 1999 and 30 September 2007., Main Outcome Measures: Hospital stay and risk of recurrence after thoracentesis versus chest tube insertion. RESULTS. Seventy-seven patients with 114 episodes of primary spontaneous pneumothorax were reviewed. They were significantly taller (P<0.001) and thinner (P<0.001) than the population mean percentile. Both the Light index and Collins formula were accurate in quantifying pneumothorax volume, but as the former was simpler and more user-friendly, this was more applicable in children. Thoracentesis resulted in shorter hospital stays (mean, 4.6; standard deviation, 1.9 days) than chest tube insertion (6.9; 3.0 days), but there was no significant difference in the recurrence rates within 6 months (P=1.0), 1 year (P=0.9), and 2 years (P=0.1). Insignificant pneumothorax was treated with observation alone in 16% of the patients. For patients with a clinically significant pneumothorax, thoracentesis and chest tube insertion were successful in 78% and 67%, respectively (P=0.34). The success rate of video-assisted thoracoscopic surgery was 89%, and postoperative recurrence occurred more commonly in patients without a lung bleb., Conclusion: Chinese children with primary spontaneous pneumothorax exhibited similar demographic characteristics to Caucasian children. Light index is simple and accurate for quantifying pneumothorax volume in children. Conservative treatment including observation, thoracentesis, and chest tube insertion should suffice for most patients with first episode of primary spontaneous pneumothorax. Early surgery is warranted for any patient who fails conservative treatment, for which video-assisted thoracoscopic surgery is safe and effective.

Monodisperse microspheres of reconstituted silkworm cocoon silk were produced using a glass capillary-based microfluidic system and by identifying an appropriate solvent/nonsolvent fluid system. The microspheres can be produced to a range of different diameters depending on the system flow rates and have a nearly homogeneous size distribution. The silk microspheres exhibit a unique core--shell architecture and have a largely beta-sheet structure, as measured by infrared spectroscopy. Mechanical characterization was performed with AFM nanoindentation and indicates that the microspheres are unexpectedly soft for a silk material. Because silk is well established as biocompatible and biodegradable, we anticipate that these silk microspheres could have particular utility in drug delivery and controlled release.

Gap junctions are protein channels between cells that allow direct electrical and metabolic coupling via the exchange of biomolecules and ions. Their expression, though ubiquitous in most mammalian cell types, is especially important for the proper functioning of cardiac and neuronal systems. Many existing methods for studying gap junction communication suffer from either unquantifiable data or difficulty of use. Here, we measure the extent of dye spread and effective diffusivities through gap junction connected cells using a quantitative microfluidic cell biology platform. After loading dye by hydrodynamic focusing of calcein/AM, dye transfer dynamics into neighboring, unexposed cells can be monitored via timelapse fluorescent microscopy. By using a selective microfluidic dye loading over a confluent layer of cells, we found that high expression of gap junctions in C6 cells transmits calcein across the monolayer with an effective diffusivity of 3.4 x 10(-13) m(2)/s, which are highly coupled by Cx43. We also found that the gap junction blocker 18alpha-GA works poorly in the presence of serum even at high concentrations (50 microM); however, it is highly effective down to 2.5 microM in the absence of serum. Furthermore, when the drug is washed out, dye spread resumes rapidly within 1 min for all doses, indicating the drug does not affect transcriptional regulation of connexins in these Cx43+ cells, in contrast to previous studies. This integrated microfluidic platform enables the in situ monitoring of gap junction communication, yielding dynamic information about intercellular molecular transfer and pharmacological inhibition and recovery.

When measuring the dielectric properties of aqueous samples, the impedance of the electrode/sample interface can limit low frequency measurements. The electrode polarization problem can be reduced by increasing the effective surface area of the electrodes. In this work, impedance spectroscopy was used to characterize and compare three different electrode surfaces that can be used to mitigate this effect: platinum black, iridium oxide, and [polypyrrole/poly(styrenesulphonate)] (PPy/PSS) conducting polymer. All three materials were directly compared with a bright platinum electrode. Equivalent circuit models were used to extract the increase in the effective surface area of the electrodes: platinum black, iridium oxide and PPy/PSS increase the effective capacitance of the electrode by factors of approximately 240, 75, and 790, respectively. The practical aspects of all electrode materials are discussed. These results suggest that iridium oxide and PPy/PSS are good alternatives to the commonly used platinum black, which is prone to mechanical damage (scratches) and is potentially toxic to cells.

In this paper, we present a method combining surface-enhanced Raman scattering (SERS) spectroscopy to detect biomolecules in a label-free way with an electrokinetic preconcentration technique (electrophoresis) to amplify biomolecular signals at low concentrations. A constant electric field is applied to charged biomolecules in solution, attracting them to an oppositely charged electrode, which is also used as a SERS substrate. Within 5 min, we observed that the SERS signal of 10 fM adenine was amplified to the level of the signal of non-preconcentrated 1 microM adenine (sensitivity improvement by 8 orders of magnitude) and the method was effective over a wide range of concentrations (10 fM to 1 microM). The signals were further amplified under stronger electric field and longer application: The increase of the signal intensity was observed to be 51 times at -0.6 V cm(-1) after 25 min. The effectiveness of this method allows the creation of label-free, target-specific, and highly sensitive monitoring applications.

We present a systematic theoretical study of core-satellite gold nanoparticle assemblies using the Generalized Multiparticle Mie formalism. We consider the importance of satellite number, satellite radius, the core radius, and the satellite distance, and we present approaches to optimize spectral shift due to satellite attachment or release. This provides clear strategies for improving the sensitivity and signal-to-noise ratio for molecular detection, enabling simple colorimetric assays. We quantify the performance of these strategies by introducing a figure of merit. In addition, we provide an improved understanding of the nanoplasmonic interactions that govern the optical response of core-satellite nanoassemblies.

Highly selective and sensitive optical methods for the detection of metal ions have had a substantial impact on molecular biology, environmental monitoring and other areas of research. Here we demonstrate a new method for detecting metal ions that is based on selective plasmonic resonance energy transfer (PRET) between conjugated metal-ligand complexes and a single gold nanoplasmonic probe. In addition to offering high spatial resolution due to the small size of the probe, our method is 100 to 1,000 times more sensitive than organic reporter-based methods. Moreover, it can achieve high selectivity owing to the selective formation of Cu(2+) complexes and selective resonant quenching of the gold nanoplasmonic probe by the conjugated complexes. We expect that PRET-based metal ion sensing could have applications in cellular imaging, systems biology and environmental monitoring.

Precisely constructed nanoscale devices and nanoarchitectures with high spatial resolution are critically needed for applications in high-speed electronics, high-density memory, efficient solar cells, optoelectronics, plasmonics, optical antennas, chemical sensors, biological sensors, and nanospectroscopic imaging. Current methods of classical optical lithography are limited by the diffraction effect of light for nanolithography, and the state of art of e-beam or focused ion beam lithography limit the throughput and further reduction less than few nanometers for large-area batch fabrication. However, these limits can be surpassed surprisingly by utilizing the overlap of two shadow images. Here we present shadow overlap of ion-beam lithography (SOIL), which can combine the advantages of parallel processing, tunable capability of geometries, cost-effective method, and high spatial resolution nanofabrication technique. The SOIL method relies on the overlap of shadows created by the directional metal deposition and etching angles on prepatterned structures. Consequently, highly tunable patterns can be obtained. As examples, unprecedented nanoarchitectures for optical antennas are demonstrated by SOIL. We expect that SOIL can have a significant impact not only on nanoscale devices, but also large-scale (i.e., micro and macro) three-dimensional innovative lithography.

Biologically functional cationic phospholipid-gold nanoplasmonic carriers have been designed to simultaneously exhibit carrier capabilities, demonstrate improved colloidal stability, and show no cytotoxicity under physiological conditions. Cargo, such as RNA, DNA, proteins, or drugs, can be adsorbed onto or incorporated into the cationic phospholipid bilayer membrane. These carriers are able to retain their unique nanoscale optical properties under physiological conditions, making them particularly useful in a wide range of imaging, therapeutic, and gene delivery applications that utilize selective nanoplasmonic properties.

We present the first optical study of large-area random arrays of crescent-shaped nanoholes. The crescent-shaped nanohole antennae, fabricated using wafer-scale nanosphere lithography, provide a complement to crescent-shaped nanostructures, called nanocrescents, which have been established as powerful plasmonic biosensors. With both systematic experimental and computational analysis, we characterize the optical properties of crescent-shaped nanohole antennae and demonstrate tunability of their optical response by varying all key geometric parameters. Crescent-shaped nanoholes have reproducible sub-10-nm tips and are sharper than corresponding nanocrescents, resulting in higher local field enhancement, which is predicted to be |E|/|E(0)| = 1500. In addition, the crescent-shaped nanohole hole-based geometry offers increased integratability and the potential to nanoconfine analyte in "hot-spot" regions, increasing biomolecular sensitivity and allowing localized nanoscale optical control of biological functions.

We propose a new solution for high hot-spot density creation by coupling a particle and a cavity in a structure dubbed a plasmonic enhanced particle-cavity (PEP-C) antenna. In comparison to analogous particle-based dimer antenna structures, the PEP-C allows both a higher maximum field and an order-of-magnitude higher hot-spot density. In addition, the hot-spots of the PEP-C antenna can be precisely controlled, resulting in increased reliability. We elucidate the photonic characteristics of the PEP-C antenna and show tuning and optimization through choice of geometric parameters. These properties make the PEP-C antenna an excellent candidate for plasmonic-based biomolecular sensors.

With a systematic comparison of the near- and far-field measures of plasmon resonance, we show that significant differences arise between the measures for both gold and silver spherical particles. The difference of the peak wavelengths between the near- and far-field measures increases with increasing particle size, reaching over 200 nm for a particle radius of 100 nm for both gold and silver. We physically explain these results by applying radiation damping to the quasi-static approximation, and we provide simple phenomenonological fits, which readily convert between the peak wavelengths for each measure. We expect that taking into account these differences can provide improvement in understanding and optimizing surface-enhanced spectroscopies.

Micropatterning is a powerful technique to custom-make and precisely control the surface topography of materials, which is determinant for a better interaction with cells. A modification of conventional micropatterning is proposed here to fabricate textured film from stiff and sticky polymers such as poly(lactide(s)-co-glycolide(s)) (PLGA) without the use of supports or solvents. Micropatterned PLGA films with square pits varying in height and channels varying in width were made to study the influence of these topographical parameters on human fibroblasts proliferation, morphology, and alignment. With increasing the square pit height, the cell attachment efficiency increased. After 10 days of culture the micropatterned films supported a significantly higher cell proliferation than smooth films. In particular, cell growth was highly stimulated in 150-mum-wide channels. Fibroblasts were spread with a typical spindle shape in all the films. Cell spreading increased with increasing the textured dimensions. A random cell organization was found for smooth and for square pit samples, and a high alignment was observed along the 150-mum-wide channels. Smaller and bigger channels did not support substantial cell growth, suggesting a possible "recognition" mechanism of the cells for optimal organization. These findings could be useful in tissue engineering applications where higher proliferation rates and eventual random or unidimensional alignments of cells are desirable., ((c) 2008 Wiley Periodicals, Inc.)

Background/aims: Hepatitis C virus (HCV) infection carries a significant risk for development of insulin resistance (IR) and/or diabetes mellitus. Recently, retinol-binding protein 4 (RBP4) has been reported as a protein contributing to IR. This study aimed to assess the correlation between RBP4 and disease severity of chronic HCV infection (CHC)., Methods: Serum RBP4 was measured in 105 treatment-nai ve CHC patients and its correlation with the homeostasis model assessment of insulin resistance index (HOMA-IR), liver histology, virology and metabolic factors was investigated. Patients were stratified into different stages of glucose tolerance by oral glucose tolerance test., Results: There was a significant decreasing linear trend of RBP4 dependent on both histological grading (from 35.8+/-16.5 microg/mL of minimal to 19.2+/-12.5 microg/mL of severe, P=0.002) and staging (from 34.2+/-10.0 microg/mL of F0 to 22.2+/-11.9 microg/mL of F3-4, P=0.02) progression, whilst a significant increment of HOMA-IR was found. Multivariate regression analysis showed BMI (1.1, 95% CI 0.44 ~ 1.77, P=0.001), HDL-C (-0.40, 95% CI -0.73 ~ -0.06, P=0.02), and LDL-C (0.31, 95% CI 0.02 ~ 0.61, P=0.04) were the significant variables for prediction of RBP4., Conclusions: Disease severity may limit the role of RBP4 as a predictor of IR in CHC.

Near infrared-absorbing gold nanoplasmonic particles (GNPs) are used as optical switches of gene interference and are remotely controlled using light. We have tuned optical switches to a wavelength where cellular photodamage is minimized. Optical switches are functionalized with double-stranded oligonucleotides. At desired times and at specific intracellular locations, remote optical excitation is used to liberate gene-interfering oligonucleotides. We demonstrate a novel gene-interfering technique offering spatial and temporal control, which is otherwise impossible using conventional gene-interfering techniques.

In this paper, signal amplifications of surface-enhanced Raman scattering (SERS) are realized by an optofluidic compact disc (CD)-based preconcentration method for effective label-free environmental and biomolecular detections. The preconcentration of target molecules is accomplished through the accumulation of adsorbed molecules on SERS-active sites by repeating a 'filling-drying' cycle of the assay solution in the optofluidic CD platform. After 30 cycles, the clear and high SERS signal of rhodamine 6G of 1 nM is readily detected. In addition to the preconcentration-based signal amplification by the optofluidic SERS system on the CD platform, we introduce a controlled precipitation of gold nanoparticles by CuSO4 for SERS substrates. This method provides high-throughput, high-sensitive and large-area uniform SERS substrates on the optofluidic CD platform. The uniform SERS signals from different positions in spots of 3 mm2 on the different CDs gives us confidence in the reliability and stability of our SERS substrates.

Spiders and silkworms employ the complex flow of highly concentrated silk solution as part of silk fiber spinning. To understand the role of fluidic forces in this process, the flow of silk solution in the spider major ampullate and silkworm silk glands was investigated using numerical simulation. Our simulations demonstrate significant differences between flow in the spider and silkworm silk glands. In particular, shear flow effects are shown to be much greater in the spider than the silkworm, the silkworm gland exhibits a much different flow extension profile than the spider gland, and the residence time within the spider gland is eight times greater than in the silkworm gland. Lastly, simulations on the effect of spinning speed on the flow of silk solution suggest that a critical extension rate is the initiating factor for fiber formation from silk solution. These results provide new insight into silk spinning processes and will guide the future development of novel fiber spinning technologies.

This paper presents satellite nanoscopes and biological application specific integrated circuits (BioASICs). Satellite nanoscope is developed for spectroscopic imaging of living cells, molecular optogenetics (i.e. the remote control of gene regulation and protein expression), and therapeutic applications. Fundamental understanding of living cells via satellite nanoscopes will provide insights of molecular dynamics and signaling pathways in living cells. BioASICs are accomplished to permit precise and repeatable characterizations of cellular activity. These BioASICs are facilitating significant advancements in quantitative cell biology and the foundation for precision biology, cell reprogramming, highspeed biologic microprocessors, drug screening, molecular diagnostics, and personalized medicine.

We describe the development of innovative plasmon resonance energy transfer (PRET)-based molecular imaging of biomolecules in living cells. Our strategy of in vivo PRET imaging relies on the resonant plasmonic energy transfer from a gold nanoplasmonic probe to conjugated target molecules, which creates "quantized quenching dips" within the Rayleigh scattering spectrum of the probe. The positions of these quantized quenching dips exactly match with the absorption peaks of the target molecule since we intentionally design nanoantennas (i.e., nanoplasmonic probes) to overlap the electronic dipoles of the molecule and the plasmonic resonance dipole of nanoantennas. Such the quenching dips allow quantitative and long-term dynamic imaging of the target molecule without the drawbacks of photobleaching and blinking inherent to fluorescent markers, which cannot provide chemical fingerprints. Compared with other imaging methods, our PRET spectroscopic imaging method allows us to generate nanoscale specific wavelengths of local light sources in living systems via nanoantennas and transmit back the nanospectroscopic imaging data of biochemical activities in living cells. As a first demonstration of in vivo PRET imaging, we performed a visualization of the dynamics of intracellular cytochrome c in HepG2 cells under ethanol-induced apoptosis.

We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 microL of crude lysate in under 30 min for the entire process.

Despite a growing focus from the academic community, the field of microfluidics has yet to produce many commercial devices for point-of-care (POC) diagnostics. One of the main reasons for this is the difficulty in producing low-cost, sensitive, and portable optical detection systems. Although electrochemical methods work well for certain applications, optical detection is generally regarded as superior and is the method most widely employed in laboratory clinical chemistry. Conventional optical systems, however, are costly, require careful alignment, and do not translate well to POC devices. Furthermore, many optical detection paradigms such as absorbance and fluorescence suffer at smaller geometries because the optical path length through the sample is shortened. This review examines the innovative techniques which have recently been developed to address these issues. We highlight microfluidic diagnostic systems which demonstrate practical integration of sample preparation, analyte enrichment, and optical detection. We also examine several emerging detection paradigms involving nanoengineered materials which do not suffer from the same miniaturization disadvantages as conventional measurements.

We describe an aptamer-based surface enhanced resonance Raman scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human alpha-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single-step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5'-capped, 3'-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes.

Unlabelled: We examined the prevalence of medical problems in children and teenagers with Down syndrome in Hong Kong., Methods: Children with Down syndrome receiving care from seven regional hospitals were included and their hospital records were reviewed. A total of 407 patients, aged between 0.06 and 17.16 years were included. Cardiovascular problems were observed in 216 (53%), endocrine problems in 111 (27%), gastrointestinal problems in 46 (11%), haematological problems in 18 (4%), neurological problems in 27 (7%), sleep problems in 36 (9%), skeletal problems in 56 (14%), visual problems in 195 (48%) and auditory problems in 137 (34%)., Conclusions: The prevalence of medical problems was high in children and teenagers with Down syndrome in Hong Kong and similar to previous findings elsewhere. Future studies on the local prevalence of medical problems in the adult population with Down syndrome would help to define their medical needs.

We present an on-chip microfluidic sample concentrator and detection triggering system for microparticles based on a combination of insulator-based dielectrophoresis (iDEP) and electrical impedance measurement. This platform operates by first using iDEP to selectively concentrate microparticles of interest based on their electrical and physiological characteristics in a primary fluidic channel; the concentrated microparticles are then directed into a side channel configured for particle detection using electrical impedance measurements with embedded electrodes. This is the first study showing iDEP concentration with subsequent sample diversion down an analysis channel and is the first to demonstrate iDEP in the presence of pressure driven flow. Experimental results demonstrating the capabilities of this platform were obtained using polystyrene microspheres and Bacillus subtilis spores. The feasibility of selective iDEP trapping and impedance detection of these particles was demonstrated. The system is intended for use as a front-end unit that can be easily paired with multiple biodetection/bioidentification systems. This platform is envisioned to act as a decision-making component to determine if confirmatory downstream identification assays are required. Without a front end component that triggers downstream analysis only when necessary, bio-identification systems (based on current analytical technologies such as PCR and immunoassays) may incur prohibitively high costs to operate due to continuous consumption of expensive reagents.

The production of transforming growth factor beta 1 (TGF-beta1) has been reported as being significantly associated with the gene polymorphism in the leader sequence at positions +29. The current study aimed to evaluate the associations between the polymorphism and the clinical characteristics of chronic hepatitis C (CHC). A total of 422 (252 men; mean age: 49.7 +/- 11.2 years) Taiwanese CHC patients with liver biopsies were enrolled. The TGF-beta1 gene polymorphism at position +29 (T or C), hepatitis C virus (HCV) RNA genotypes, and serum HCV RNA levels of these patients were determined. Of the 422 patients, the frequency of the T allele was 45.4%. Based on univariate analyses, a significantly lesser proportion of patients with allele T had high viral loads than those who were without allele T (P = 0.026). The lesser HCV RNA levels and HCV genotype 1b infection were significantly associated with the inheritance of the T allele in female patients based on univariate (P = 0.012 and 0.007, respectively) and multivariate regression (odds ratio/95% confidence interval: 0.434/0.219-0.859 and 0.468/0.237-0.927, respectively) analyses. In male patients with or without inheritance of the T allele, the clinical characteristics were similar. In conclusion, the association between TGF-beta1 polymorphism and virologic characteristics of chronic HCV infection implicated a significant role of host genetic factors on the clinical features of CHC. Female patients who carry T allele at position +29 were predisposed to be associated with HCV genotype non-1b infection and lesser HCV viral load, which revealed the gender effect.

The prevalence of hepatitis C virus (HCV) infection among adults in aboriginal areas has been shown to be higher than in urban areas in Taiwan. Whether the prevalence of HCV infection is also higher among children in aboriginal areas remains unclear. In total, 1176 schoolchildren in four aboriginal areas were invited to participate in the study. All children were tested for serum antibodies to HCV (anti-HCV) and liver enzymes. The age range of children was 6-13 years. Another 606 sex- and age-matched schoolchildren from an urban area served as controls. There was no statistically significant difference in prevalence of anti-HCV between aboriginal and Han Chinese students in aboriginal areas. The prevalence of anti-HCV was 0.3% (4/1176) in aboriginal areas, which was similar to the prevalence of 0% (0/606) in the urban area. The four anti-HCV seropositive aboriginal children were all negative for HCV RNA. Our data suggest that the high prevalence of anti-HCV among aboriginal adults might be due to subsequent exposure to risk factors after school age.

Tzukuan Township in Taiwan has been reported to be an endemic area for hepatitis C virus (HCV) infection both in adults and adolescents. The maritime part of the township carries a higher prevalence than the non-maritime part and, as a consequence, several public education strategies have been introduced during the past decade. The current follow-up study aimed to clarify the changing prevalence of HCV infection among teenagers in the endemic maritime part of Tzukuan. In addition to viral hepatitis markers and biochemical profiles, we compared the epidemiological characteristics of 887 and 394 teenagers (aged 13-16 years) from the maritime part enrolled in 1995 and 2005, respectively. Compared with the results of surveillance in 1995, the prevalence of anti-HCV seropositivity (1.0% vs. 2.8%; P=0.045) and HCV RNA (0.5% vs. 2.3%; P=0.026) had decreased significantly by 2005. Transfusions and anti-HCV-positive families were the main risk factors amongst the 25 anti-HCV-positive teenagers in 1995, and became non-significant amongst the four anti-HCV-positive teenagers in 2005. In conclusion, the seroprevalence of HCV infection has significantly decreased after one decade of intervention among the teenage population in this endemic area.

Objectives: There is growing evidence suggesting the mutual link between type 2 diabetes mellitus (T2DM) and hepatitis C virus (HCV) infection. However, the impact of HCV infection on the suite of glucose abnormalities has rarely been investigated. The study aimed to determine the difference regarding the prevalence and the characteristics of glucose abnormalities between chronic hepatitis C (CHC) patients and community-based controls. It also aimed to investigate the related clinical, virological, and histological features of glucose abnormalities in HCV infection., Methods: Six hundred eighty-three CHC patients and 515 sex-/age-matched controls were included. Oral glucose tolerance test (OGTT) was performed in 522 CHC patients and 447 controls without known T2DM. Clinical data were assessed upon the different stages of glucose abnormalities based on OGTT results., Results: The prevalence of normoglycemia, IGT, and T2DM in 683 CHC patients was 27.7%, 34.6%, and 37.8%, respectively. There was a significant linear trend from normoglycemia to T2DM in terms of age, family history of T2DM, and advanced liver fibrosis in CHC patients. For those CHC patients without fibrosis, the prevalence of glucose abnormalities reached 67.9% high. All CHC patients carried a significantly higher prevalence than controls regarding those aged <65 yr. For those without known DM, there was a 3.5-fold increase in the prevalence of glucose abnormalities in CHC (65.8%) patients in comparison with controls (35.3%) (OR 3.51, 95% CI 2.70-4.56, P < 0.001)., Conclusions: CHC patients carried a high prevalence of glucose abnormalities. Determination of glucose abnormalities by OGTT may be suggested.

We present a systematic numerical study of plasmon resonance of the nanocrescent. We show that by varying the nanocrescent geometry, the plasmon resonance peak can be tuned into the near-infrared and local field enhancement can be increased significantly, with maximum enhancement of the electric field amplitude of approximately 100 for realistic geometric parameters. Because of its wide tunability, high local field enhancement, and geometry which utilizes both sharp features and intra-particle coupling, the nanocrescent is a structure well suited for in vivo cellular imaging as well as in vitro diagnostic applications.

Background/aims: To evaluate the association of virologic status with serum cholesterol and triglyceride levels in individuals with hepatitis C virus (HCV) infection., Methods: We conducted a large scale community-based study enrolling 11,239 residents in an area endemic for hepatitis B virus (HBV) and HCV infection in southern Taiwan. Overall, 703 (6.3%), 1,536 (13.7%), 84 (0.7%) and 9,084 (80.8%) subjects were sero-positive for anti-HCV antibody (anti-HCV), hepatitis B surface antigen (HBsAg), and both anti-HCV and HBsAg, and negative for anti-HCV and HBsAg, respectively., Results: By multivariate logistic analyses, the independent factors significantly associated with elevated serum cholesterol level were older age, female, negative for diabetes, anti-HCV or HBsAg and elevated triglyceride levels. The independent factors significantly associated with elevated serum triglyceride level were male, positive for diabetes, negative for anti-HCV or HBsAg, higher body mass index (BMI) and elevated cholesterol levels. Of 642 anti-HCV-positive subjects that have HCV RNA tested by standardized automated qualitative PCR assay, 478 (74.5%) were positive for HCV RNA. By multivariate logistic analyses, the independent factors associated with elevated serum cholesterol level were female, elevated serum triglyceride levels, negative for diabetes or HCV RNA. The independent factors associated with elevated serum triglyceride levels were elevated serum cholesterol levels, positive for diabetes, higher BMI and negative for HCV RNA. Diabetes, lower cholesterol and triglyceride levels were independent factors associated with positive HCV RNA., Conclusions: Based on the result of this large scale community study, HCV viremia appears to be associated with lower serum cholesterol and triglyceride levels which implies that HCV itself might play a significant role on serum lipid profile of patients with chronic HCV infection.

We report on integrated optofluidic Raman-activated cell sorting (RACS) platforms that combine multichannel microfluidic devices and laser tweezers Raman spectroscopy (LTRS) for delivery, identification, and simultaneous sorting of individual cells. The system allows label-free cell identification based on Raman spectroscopy and automated continuous cell sorting. Two optofluidic designs using hydrodynamic focusing and pinch-flow fractionation are evaluated based on their sorting design and flow velocity effect on the laser trapping efficiency at different laser power levels. A proof-of-principle demonstration of the integrated optofluidic LTRS system for the identification and sorting of two leukemia cell lines is presented. This functional prototype lays the foundation for the development of a label-free cell sorting platform based on intrinsic Raman markers for automated sampling and sorting of a large number of individual cells in solution.

Unlabelled: Recommended treatment for hepatitis C virus genotype 1 (HCV-1) patients is peginterferon plus ribavirin for 48 weeks. We assessed whether treatment duration of 24 weeks is as effective as standard treatment in HCV-1 patients with a rapid virological response (RVR; seronegative for hepatitis C virus [HCV] RNA at 4 weeks). Two hundred HCV-1 patients were randomized (1:1) to either 24 or 48 weeks of peginterferon-alpha-2a (180 microg/week) and ribavirin (1000-1200 mg/day) with a 24-week follow-up. The primary endpoint was a sustained virological response (SVR; seronegative for HCV RNA at 24-week follow-up). Overall, the 48-week arm had a significantly higher SVR rate (79%) than the 24-week arm (59%, P = 0.002). For 87 (43.5%) patients with an RVR, the 24-week arm had a lower SVR rate [88.9%; 95% confidence interval (CI): 80%-98%] than the 48-week arm (100%, P = 0.056). For 52 patients with low baseline viremia (<400,000 IU/mL) and an RVR, the 24-week arm had rates (CI) of relapse and SVR of 3.6% (-3%-11%) and 96.4% (89%-103%), respectively, which were comparable to those of the 48-week arm (0% and 100%) with difference (CI) of 3.6% (-7.2%-6.6%) and -3.6% (-14.3% to -0.6%), respectively. Multivariate analysis in all patients showed that RVR was the strongest independent factor associated with an SVR, followed by treatment duration, mean weight-based exposure of ribavirin, and baseline viral load., Conclusion: HCV-1 patients derive a significantly better SVR from 48 weeks versus 24 weeks of peginterferon/ribavirin even if they attain an RVR. Both 24 and 48 weeks of therapy can achieve high SVR rates (>96%) in HCV-1 patients with low viral loads and an RVR.

13C cross-polarization magic angle spinning (CPMAS) NMR spectroscopy is demonstrated to be a valuable characterization tool for quantitative measurements of Hg2+ adsorption capacity in thiol-functionalized mesoporous silica SBA-1 (Santa Barbara Amorphous-1). This is the first report on the investigation of the spectral change in the 13C CP signals for mercaptopropyl-functionalized mesoporous materials doped with different Hg2+ concentrations. The chemical shift of the carbon atom adjacent to the thiol group is sensitive to the binding of the mercury ion, and its peak intensity can be used as a quantitative sensor for the amount of the mercury ion adsorbed. The 13C CPMAS NMR results are in good agreement with inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analysis.

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of in vivo tumors. Moreover, continuous dynamic perfusion allows the establishment of physiologically relevant drug profiles to exposed spheroids. Here we present a physiologically inspired design allowing microfluidic self-assembly of spheroids, formation of uniform spheroid arrays, and characterizations of spheroid dynamics all in one platform. Our microfluidic device is based on hydrodynamic trapping of cancer cells in controlled geometries and the formation of spheroids is enhanced by maintaining compact groups of the trapped cells due to continuous perfusion. It was found that spheroid formation speed (average of 7 h) and size uniformity increased with increased flow rate (up to 10 microl min(-1)). A large amount of tumor spheroids (7,500 spheroids per square centimeter) with a narrow size distribution (10 +/- 1 cells per spheroid) can be formed in the device to provide a good platform for anticancer drug assays.

We present a novel approach for the ultra-rapid direct patterning of complex three-dimensional, stacked polystyrene (PS) microfluidic chips. By leveraging the inherent shrinkage properties of biaxially pre-stressed thermoplastic sheets, microfluidic channels become thinner and deeper upon heating. Design conception to fully functional chips can thus be completed within minutes.

Aims: Positive serum antinuclear antibody (ANA) is present in a number of patients with chronic hepatitis C virus (HCV) infection. This study aimed to evaluate the prevalence of ANA in patients with chronic hepatitis C (CHC) and to elucidate its clinical implications in virological and histological characteristics of CHC infection., Methods: A total of 614 CHC patients were enrolled in this prospective, hospital-based study. The serum levels of aspartate aminotransferase, alanine aminotransferase and ANA, and HCV genotype, HCV RNA level, and histological activity index scores for liver histopathology, were determined., Results: The prevalence of positive ANA (titre >1:40) was 35.0%. Women had a significantly higher prevalence than men (41.2 vs 31.0%; p = 0.012). Patients positive for ANA were significantly older (mean (SD), 53.7 (10.5) vs 49.7 (11.3) years; p<0.001) and had higher mean (SD) alanine aminotransferase levels (186.9 (178.8) vs 155.50 (113.5) IU/l; p<0.001) and lower mean (SD) HCV RNA levels (5.2 (0.9) vs 5.4 (1.0) log IU/ml; p = 0.048) than those without ANA. Among 447 patients undergoing liver biopsy, those positive for ANA had a significantly higher mean (SD) fibrosis score (2.0 (1.3) vs 1.5 (1.1); p<0.001) and a higher frequency of F3-4 (69/187, 36.9% vs 50/260, 19.2%; p<0.001) than those negative for ANA. Multivariate logistic regression analyses showed that advanced fibrosis, lower HCV RNA levels and age were significant factors related to positive ANA., Conclusion: ANA is associated with a more advanced liver fibrosis and lower serum HCV RNA level in patients with CHC.

We present a microfluidic bioreactor for culturing high-density arrays of hepatocytes in a tissue-like micro-architecture. The microfluidic environment mimicked physiological liver mass transport, enabling sustained culture of high density cells (>2,000 cells/mm(2)) without nutrient limitation for over 1 week. The key feature of this design was a microporous microfluidic barrier that formed a sieved-pocket to concentrate cells during loading. Nutrient depletion within the cell mass was avoided by maintaining a continuous flow of medium (10 microl/day) that diffused across the porous barrier. Human hepatoma cells (HepG2/C3A) remained viable and functional as demonstrated by fluorescent viability assays and secretion of albumin for the one-week culture period.

Pantothenate kinase-associated neurodegeneration (formerly Hallervorden-Spatz syndrome), the most prevalent form of neurodegeneration with brain iron accumulation, is a rare degenerative brain disease characterised by predominantly extrapyramidal dysfunction resulting from mutations in the PANK2 (pantothenate kinase 2) gene. Using DNA mutation analysis, the authors identified a novel missense mutation (P354L) in exon 4 of the PANK2 gene in an adolescent with classic pantothenate kinase-associated neurodegeneration. DNA-based diagnosis of pantothenate kinase-associated neurodegeneration plays a key role in determination, and can make the diagnosis more simply, directly, and economically because it obviates the need for unnecessary biochemical tests. Once pantothenate kinase-associated neurodegeneration-like symptoms are identified, mutation analysis and target screening for the family of the proband can provide efficient and accurate evidence of pantothenate kinase-associated neurodegeneration inheritance.

We present a rapid and non-photolithographic approach to microfluidic pattern generation by leveraging the inherent shrinkage properties of biaxially oriented polystyrene thermoplastic sheets. This novel approach yields channels deep enough for mammalian cell assays, with demonstrated heights up to 80 microm. Moreover, we can consistently and easily achieve rounded channels, multi-height channels, and channels as thin as 65 microm in width. Finally, we demonstrate the utility of this simple microfabrication approach by fabricating a functional gradient generator. The whole process--from device design conception to working device--can be completed within minutes.

Background: Polymerase chain reaction (PCR) methods play an essential role in providing data relating to diagnosis, monitoring and treatment of hepatitis C virus (HCV) infection. The real-time reverse transcription PCR (RT-PCR) assay is an established and promising tool in terms of quantifying HCV RNA for clinical application. This study aimed to evaluate the performance characteristics of a real-time RT-PCR-based test in a clinical setting., Methods: Validation and reproducibility tests were performed using a standard panel. Sera from 197 chronic HCV patients were analyzed by the real-time RT-PCR assay and the results were compared with the Versant bDNA3.0 assay (bDNA3.0)., Results: The real-time RT-PCR assay showed an acceptable linear response (r2=0.989-0.995) in the serial dilutions regarding genotypes 1b, 2a, 2b and 1b+2a. HCV viral loads were quantifiable in all 197 patients (100%) by the real-time RT-PCR assay and in 194 (98.5%) by the bDNA3.0. HCV RNA quantification values measured by the real-time RT-PCR and bDNA3.0 assays were positively correlated (Pearson's correlation coefficient r=0.734, p<0.001). The real-time RT-PCR assay values were on average 0.13 logs higher than the bDNA3.0 results. The correlation coefficients with genotypes 1b, 2a, 2b and mixed were 0.737, 0.711, 0.791 and 0.766, respectively (p<0.01)., Conclusions: The real-time RT-PCR assay showed comparable performance with bDNA3.0 regarding quantification of HCV viral loads with genotype 1 and 2 HCV infections.

This study aimed to elucidate the rate and predictors of early (6 months) therapeutic responses to lamivudine, the rate of early mortality and the use of the model for end-stage liver disease (MELD) and Index in predicting the survival in patients with a clinical diagnosis of non-cirrhotic chronic hepatitis B with decompensation. Ninety-eight patients with lamivudine therapy were enrolled and MELD and Index scores were calculated. Surviving patients were treated with lamivudine for more than 6 months. Four (4.1%) of the 98 patients died after initiation of lamivudine therapy. After a 6-month lamivudine therapy, 80 (85.1%) patients and 71 (75.5%) patients had normal alanine aminotransferase (ALT) values and negative hepatitis B virus (HBV) DNA (<200 copies/mL), respectively, and hepatitis B e antigen (HBeAg)-negative patients had a significantly higher rate of negative HBV DNA than HBeAg-positive patients (P=0.002). The rates of HBeAg seroconversion and negative HBV DNA were 28.8 and 63.5%, respectively, and patients with HBeAg seroconversion had a significantly higher rate of negative HBV DNA (P=0.004). By multivariate analyses, older age, HBV nongenotype B infection, negative HBeAg and higher ALT levels were factors associated with negative HBV DNA, and a higher ALT level was associated with HBeAg seroconversion at month 6 after lamivudine therapy. MELD score and Index score were significantly associated with death and areas under the receiver operating characteristic curve for predicting survival were 0.936 and 0.907 respectively. We concluded that after 6-month lamivudine therapy, the patients who survived achieved favourable biochemical, virological responses and rate of HBeAg seroconversion. Both MELD and Index scoring systems are good models to predict the 6-month survival.