Using poly(3-aminophenylboronic acid) thin film with binding-induced ion flux blocking for amperometric detection of hemoglobin A1c.

This study reports a novel enzyme-free, label-free amperometric method for direct detection of hemoglobin A1c (Hb(A1c)), a potent biomarker for diabetes diagnosis and prognosis. The method relies on an electrode modified with poly(3-aminophenylboronic acid) (PAPBA) nanoparticles (20-50 nm) and a sensing scheme named "binding-induced ion flux blocking." The PAPBA nanoparticles were characterized by FT-IR, XPS, TEM, and SEM. Being a polyaniline derivative, PAPBA showed an ion-dependent redox behavior, in which insertion or extraction of ions into or out of PABPA occurred for charge balance during the electron transfer process. The polymer allowed Hb(A1c) selectively bound to its surface via forming the cis-diol linkage between the boronic acid and sugar moieties. Voltammetric analyses showed that Hb(A1c) binding decreased the redox current of PAPBA; however, the binding did not alter the redox potentials and the apparent diffusivities of ions. This suggests that the redox current of PAPBA decreased due to an Hb(A1c) binding-induced ion flux blocking mechanism, which was then verified and characterized through an in situ electrochemical quartz crystal microbalance (EQCM) study. Assay with Hb(A1c) by differential pulse voltammetry (DPV) indicates that the peak current of a PAPBA electrode has a linear dependence on the logarithm of Hb(A1c) concentration ranging from 0.975 to 156 µM. The Hb(A1c) assay also showed high selectivity against ascorbic acid, dopamine, uric acid, glucose and bovine serum albumin. This study has demonstrated a new method for developing an electrochemical Hb(A1c) biosensor and can be extended to other label-free, indicator-free protein biosensors based on a similar redox polymer electrode.

Real-time telemetry system for amperometric and potentiometric electrochemical sensors.

A real-time telemetry system, which consists of readout circuits, an analog-to-digital converter (ADC), a microcontroller unit (MCU), a graphical user interface (GUI), and a radio frequency (RF) transceiver, is proposed for amperometric and potentiometric electrochemical sensors. By integrating the proposed system with the electrochemical sensors, analyte detection can be conveniently performed. The data is displayed in real-time on a GUI and optionally uploaded to a database via the Internet, allowing it to be accessed remotely. An MCU was implemented using a field programmable gate array (FPGA) to filter noise, transmit data, and provide control over peripheral devices to reduce power consumption, which in sleep mode is 70 mW lower than in operating mode. The readout circuits, which were implemented in the TSMC 0.18-µm CMOS process, include a potentiostat and an instrumentation amplifier (IA). The measurement results show that the proposed potentiostat has a detectable current range of 1 nA to 100 µA, and linearity with an R2 value of 0.99998 in each measured current range. The proposed IA has a common-mode rejection ratio (CMRR) greater than 90 dB. The proposed system was integrated with a potentiometric pH sensor and an amperometric nitrite sensor for in vitro experiments. The proposed system has high linearity (an R2 value greater than 0.99 was obtained in each experiment), a small size of 5.6 cm × 8.7 cm, high portability, and high integration.

Elevated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells.

BACKGROUND: The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefitinib efflux from cells. In addition, BCRP/ABCG2 expression correlates with poor response to gefitinib in both cancer cell lines and lung cancer patients with wtEGFR. Co-treatment with BCRP/ABCG2 inhibitors enhanced the anti-tumor activity of gefitinib. CONCLUSIONS/SIGNIFICANCE: Thus, BCRP/ABCG2 expression may be a predictor for poor efficacy of gefitinib treatment, and targeting BCRP/ABCG2 may broaden the use of gefitinib in patients with wtEGFR.

Electrochemical characterisation of a conductive polymer molecularly imprinted with an Amadori compound.

Type II diabetes is a disease that is often characterised by elevated levels of advanced glycation end-products (AGEs), e.g. glycated haemoglobin (HbA(1c)), in a patient's bloodstream. This glycation reaction occurs when the carbonyl group of a circulating sugar (glucose) reacts with the amino group of the terminal valine residue of a haemoglobin chain, to form an unstable imine. This compound then undergoes an Amadori rearrangement to form the stable Amadori compound N-(1-deoxy-beta-D-fructopyranose-1-yl)-L-valine (Fru-Val). As an initial approach to fabricating a sensor for the Fru-Val component of HbA(1c), molecular imprints of Fru-Val were made in poly-aminophenylboronic acid (p-APBA), using ammonium persulphate as the initiator, on conductive indium-doped tin oxide (ITO) electrodes (nominal working area 0.5 cm(2)). The affinity of the imprints formed in the p-APBA, for fructose and valine as individual molecules, as well as for the complete template used for imprinting (Fru-Val), was assessed electrochemically, by the use of open circuit potential (DeltaE(oc)) measurements. These showed that the imprinted materials when challenged with Fru-Val had an open circuit response of approximately 5.0x10(-3) V. D-fructose (10 mM), a component of the template, when introduced into the cell gave a far more significant change in the open circuit potential (DeltaE(oc)= approximately 2.9x10(-3) V) than did a similar concentration of d-glucose, a non-template carbohydrate (DeltaE(oc)= approximately 4.0x10(-4) V). Non-template structured p-APBA films, made as controls in the absence of Fru-Val, showed negligible response to either D-fructose or D-glucose. Additionally, we have shown that the imprinted films show a progressive reduction in response to sequential additions of D-fructose, implying the saturation of imprinted sites and a limit to non-specific recognition.

A systematic approach to forming micro-contact imprints of creatine kinase.

A systematic approach has been used to form molecular imprints of creatine kinase (CK) using micro-contact imprinting. Using thermocalorimetry data, we selected poly(ethylene glycol) 400 dimethacrylate (PEG400DMA) as our crosslinker, on the basis that it would be expected to have minimal specific recognition when incorporated into the imprinted polymer. The functional monomer used, methacrylic acid (MAA), was chosen from a panel of six candidates on the basis of it giving the highest differential affinity with respect to a non-imprinted polymer. A polymer formed with 5% MAA and 95% PEG400DMA showed excellent imprint recognition, with CK binding to the imprinted material being 2.05 +/- 0.07 x 10(-10) mol cm(-2) compared to 9.1 +/- 4.5 x 10(-12) mol cm(-2) control binding. The imprinted polymers (approximate thickness 22.6 mum as measured by Alpha-step) showed clear two-phase binding with maximum absorption achieved after approximately 2 hours. Data extracted from Scatchard plots showed the K(d) for the high affinity binding site population to be 2.56 x 10(-10) M and the binding site population to be 1.97 x 10(-10) mol cm(-2), corresponding data for low affinity binding sites shows the K(d) = 3.27 x 10(-9) M and the binding site population to be 2.32 x 10(-10) mol cm(-2). Re-binding the molecularly imprinted polymers (MIPs) with non-template proteins, namely myoglobin, human serum albumin (HSA) and immunoglobulin G (Ig G), showed these proteins to have comparatively little affinity for the CK imprinted films. The percentage re-binding figures, relative to CK binding, were: 18.7, 3.5, and 3.5 for myoglobin, HSA, and Ig G respectively. This pattern of binding was maintained in competitive binding protocols with two proteins in solution at equal concentrations, where the percentage re-binding figures, relative to CK binding (4.5 +/- 0.06 x 10(-10) mol cm(-2)), were 17.2, 4.5, and 2.9 for myoglobin, HSA, and Ig G respectively. The presence of multiple competing analytes in undiluted human serum did not significantly decrease template protein recognition. Finally, we used circular dichroism to monitor protein denaturation, and showed that the denatured template protein loses a significant proportion (76.8%) of its MIP affinity after being heated at 80 degrees C for 10 minutes.

Assessing the binding selectivity of molecularly imprinted polymer artificial antibodies by mass spectrometry-based profiling system.

Molecularly imprinted polymer (MIP) is a technique for generating polymer-bearing biomimetic receptors. It offers several advantages to the research such as analysis, sensors, extraction, or preconcentration of components. Myoglobin is known to be an important biological index for the diagnosis of cardiac diseases. The purpose of this research was to optimize the formation of myoglobin-imprinted polymer (Myo-MIP) and develop a mass spectrometry-based profiling system for assessing the binding selectivity of artificial antibodies formed by Myo-MIP. Experimental results showed that myoglobin and albumin were bound/absorbed onto Myo-MIP chips and not to nonimprinted polymer (NIP) chips. Other proteins, such as histidine-rich glycoprotein, immunoglobulins, proapolipoprotein, and leech-derived tryptase inhibitor, were also observed but with less reproducibility from the chips.

Integrated microfluidic system for electrochemical sensing of urinary proteins.

This study reports a new microfluidic system integrated with a microfluidic control module and a micro electrochemical module for detection of urinary proteins. The integrated microsystem can automatically detect proteins in urine with a high sensitivity. The microfluidic control module consists of a new two-way, spiral-shape micropump which can transport the urine samples to the sensing regions. The net ionic charges of the protein samples can be detected while the samples flow through the sensing region of the micro electrochemical module. Two major urinary proteins including lysozyme and albumin are detected in a multiple-channel layout with little human intervention and are analyzed in a short period of time, while only consuming a 100-mul urine sample. The developed microfluidic system could lead to a convenient, yet crucial, platform for chemical and biological detection and diagnosis.

Detection of triglyceride using an iridium nano-particle catalyst based amperometric biosensor.

The detection and quantification of triglyceride (TG) using an iridium nano-particle modified carbon based biosensor was successfully carried out in this study. The detection procedures were based on the electrochemical detection of enzymatically produced NADH. TG was hydrolyzed by lipase and the glycerol produced was catalytically oxidized by NAD-dependent glycerol dehydrogenase producing NADH in a solution containing NAD(+). Glyceryl tributyrate, a short chain triglyceride, was chosen as the substrate for the evaluation of this TG biosensor in bovine serum and human serum. A linear response to glyceryl tributyrate in the concentration range of 0 to 10 mM and a sensitivity of 7.5 nA mM(-1) in bovine serum and 7.0 nA mM(-1) in human serum were observed experimentally. The potential interference of species such as uric acid (UA) and ascorbic acid (AA) was assessed. The incorporation of a selected surfactant and an increase in the incubation temperature appeared to enhance the performance of this biosensor. The conditions for the determination of TG levels in bovine serum using this biosensor were optimized, with sunflower seed oil being used as an analyte to simulate the detection of TG in blood. The experimental results demonstrated that this iridium nano-particle modified working electrode based biosensor provided a relatively simple means for the accurate determination of TG in serum.

Optimizing the formulation of a myoglobin molecularly imprinted thin-film polymer--formed using a micro-contact imprinting method.

Thin-film myoglobin molecularly imprinted polymers have been fabricated using a micro-contact approach. By initially selecting the cross-linker on the basis of it having a minimal recognition for the template and using this as a starting point for functional monomer selection, we have produced myoglobin imprinted polymers with exceptionally high selectivities. The affinity of the polymers, for myoglobin, when prepared with a variety of different cross-linkers and no functional monomer was evaluated. Of these, tetraethylene glycol dimethacrylate (TEGDMA) exhibited the lowest affinity for the template species. Methyl methacrylate (MMA) was chosen as the functional monomer as when it was used in conjunction with TEGDMA, it exhibited maximum selectivity for the template compared to polymers made with other functional monomers. With a MMA to TEGDMA ratio of 1 to 3, the myoglobin molecularly imprinted polymer adsorbed 15.03+/-0.89 x 10(-11)mole/cm(2) of template from a 5.68 x 10(-7)M myoglobin solution, compared to 2.58+/-0.02 x 10(-11)mole/cm(2) for a polymer of similar composition, but formed in the absence of a template. Various washing conditions, using alkaline media to remove the template, were investigated. An extraction solvent comprising 2 wt.% SDS and 0.6 wt.% NaOH used at 80 degrees C for 30 min was shown to give the highest imprinting factor i.e. 5.83 with 72.82% myoglobin removal. The saturation kinetics of template binding to the thin-film MIP were examined and found to display a simple two-phase profile typical of non-cooperative binding. A Scatchard binding plot showed the dissociation constant (K(d)) for the specific binding phase to be 3.4 x 10(-7)M and the binding site capacity to be 7.24 x 10(-11)mole/cm(2). For the non-specific binding phase, K(d) was found to be 1.355 x 10(-5)M and the binding site capacity was determined as 9.62 x 10(-10)mole/cm(2). Selectivity experiments were carried out in both single protein and binary protein systems all using a total protein concentration of 5.68 x 10(-7)M. The molar ratio of adsorbed myoglobin to IgG, HSA and hemoglobin was found to 115.5, 230.9 and 2.5, respectively. While, in binary competition systems, myoglobin selectivity to IgG, HSA and hemoglobin was, respectively, 94.18, 98.21 and 61.09%. Rebinding in natural biological matrices, i.e. human serum or urine, showed the imprinted films to have significantly greater uptake than non-imprinted films. Re-binding in undiluted urine was found to be a facile process, with the imprinting factor, i.e. the ratio of MIP to NIP binding, being determined as 37.4.

A portable potentiostat for the bilirubin-specific sensor prepared from molecular imprinting.

A portable amperometric potentiostat was designed and implemented in this work. It was developed to acquisit the current signals produced from bilirubin by an electrochemical sensor. Based on an SOC-based chip, this potentiostat has the merits of moderate accuracy, small size, low cost, and high portability. The bilirubin electrode was prepared by synthesizing a thin layer of bilirubin imprinted poly(methacrylic acid-co-ethylene glycol dimethacrylate) onto the Au layer. With the molecularly imprinted polymer (MIP) film, specific detection of bilirubin was successfully achieved. The cyclic voltammogram of the electrode was measured from this assembled potentiostat. The performance from a commercial potentiostat was considered rather stable and was used as a reference to examine and evaluate the performance of the assembled potentiostat. The detected current signals by the bilirubin sensing were obtained. Linear calibration with a sensitivity of 1.344+/-0.38 microA/mg dl was achieved. Our experimental results showed that the proposed potentiostat's performance could achieve sufficient performance. The evaluation was also made from the aspects such as reset time and steady-response time. The self-assembled potentiostat thus demonstrated its ability in precise detection of bilirubin from an electrode layered with the imprinted polymer film.

Development and characterization of an all-solid-state potentiometric biosensor array microfluidic device for multiple ion analysis.

A microfluidic device with an all-solid-state potentiometric biosensor array was developed using microfabrication technology. The sensor array included a pH indicator, and potassium and calcium ion-selective microelectrodes. The pH indicator was an iridium oxide thin film modified platinum microelectrode and the iridium oxide was deposited by an electrochemical method. The potassium and calcium ion-selective microelectrodes were platinum coated with silicon rubber based ion-selective membranes with respectively potassium (valinomycin) and calcium (ETH 1001) ionophores. The detection system was integrated with a micro-pneumatic pump which can continuously drive fluids into the microchannel through sensors at flow rates ranging from 52.4 microl min(-1) to 7.67 microl min(-1). The sensor array microfluidic device showed near-Nernstian responses with slopes of 62.62 mV +/- 2.5 mV pH(-1), 53.76 mV +/- 3 mV -log[K+](-1) and 25.77 mV +/- 2 mV -log[Ca2+](-1) at 25 degrees C +/- 5 degrees C, and a linear response within the pH range of 2-10, with potassium and calcium concentrations between 0.1 M and 10(-6) M. In this study the device provided a convenient way to measure the concentration of hydrogen, potassium and calcium ions, which are important physiological parameters.

The microcontact imprinting of proteins: the effect of cross-linking monomers for lysozyme, ribonuclease A and myoglobin.

The performance of molecularly imprinted polymers (MIPs) is of interest to researchers in the field of analytical chemistry, and in the pharmaceutical and food industries. Because the choice of the functional monomer(s) plays a key role in the selectivity of a MIP, the synthesis of an effective, tight-binding MIP can be difficult and time-consuming, involving the evaluation of the binding performance of MIPs of many different compositions. In this study, we report an express method combining molecular imprinting and microcontact printing techniques to prepare a polymer thin film as an artificial antibody. In addition to the microcontact printing technique, isothermal titration of monomers to proteins stamps was investigated to screen the functional monomer for MIPs. Finally, the importance of the choice of cross-linking monomers in MIPs was studied, and these studies suggest that monomers containing an optimal length PEG spacer give higher imprinting effectiveness. Several model antigens (lysozyme, ribonuclease A and myoglobin) were adsorbed on a cover glasses that were pretreated with hexamethyldisilazane (HMDS). These protein stamps were then contacted with different monomer solutions (cross-linking monomers) on a glass slide substrate. Photopolymerization yielded the molecularly imprinted polymer. This technique, analogous to microcontact printing, allows for the rapid, parallel synthesis of MIPs of different compositions, and requires very small volumes of monomers (ca. 4 microL). The technique also avoids potential solubility problems with the molecular targets. Of several cross-linking monomers screened, tetraethyleneglycol dimethacrylate (TEGDMA) gave the most selective lysozyme binding, while polyethyleneglycol 400 dimethacrylate (PEG400DMA) were most selective for ribonuclease A and myoglobin.

Using protein templates to direct the formation of thin-film polymer surfaces.

Protein imprinted electrodes formed by the cyclic voltammetric deposition of conductive polymers, on screen-printed platinum supports, in the presence of target proteins have been fabricated. An initial layer of polypyrrole was used as a supporting polymer layer, upon which were formed two layers of polyaminophenylboronic acid. The first of these layers was non-imprinted and formed a barrier between the polypyrrole and the outer layer, which was deposited in the presence of a protein template (lysozyme or cytochrome c). After protein extraction, re-binding of the template proteins to their respective imprinted electrodes showed a distinct two-phase binding profile; whereas, binding to control polymers, made in the same way but without the addition of protein templates, showed progressive binding typical of non-specific recognition. Reductions in the observed current transmission due to bonding to the polymer surface of non-conductive protein have been used as a measure of re-binding. It was found that when challenged with 1 part per million protein in solution, the current reductions for the lysozyme and cytochrome c imprinted electrodes were 30.3 and 66.2%, respectively, compared to 4.5 and 29.9% for their respective control electrodes. All measurements carried out at -0.1 V with Ag/AgCl reference.

Amperometric protein sensor - fabricated as a polypyrrole, poly-aminophenylboronic acid bilayer.

An approach to the design of electrodes for the production of sensors, which show significant changes to the passage of current in response to the concentration of target protein molecules, is presented. Screen-printed platinum electrodes, modified with two separately applied conducting polymer layers, have been developed as a potential route to forming cheap disposable protein sensors. To achieve a heightened response for the target molecules, an initial layer of polypyrrole was formed on the electrode's surface by electro-deposition. This composite was then employed as a substrate for the subsequent electro-deposition of a relatively thin 'sensing layer' of poly-aminophenylboronic acid. Cyclic voltammetry (CV) of the prepared films revealed an excursion in the current versus potential curve in the anodic phase at approximately 0.0 to +0.2V. It was clearly shown that the introduction of proteins into the CV cell resulted in a measurable decrease in the passage of current in buffered aqueous media. Measured current reductions observed on introducing lysozyme (10ppm) into the test solution were 2.3x10(-6)A for an electrode formed with a poly-aminophenylboronic acid layer on platinum, and 1.75x10(-5)A for a composite electrode formed with poly-aminophenylboronic acid on a polypyrrole coated platinum substrate. The introduction of the competing analytes, dl adrenaline or dopamine, at concentrations typically found in human urine, had little effect on the sensor's response. Additionally, the sensing system was able to maintain a response to added target proteins with as much as 2vol.% urine in the test solution. Using the electrodes in high concentrations of competing physiological analytes, they were able to respond to protein concentrations as low as 0.5ppm in buffered solutions containing urea at a concentration representative of human urine (17,000ppm), which additionally contained glucose (1000ppm).

Incorporation of styrene enhances recognition of ribonuclease A by molecularly imprinted polymers.

Ribonuclease A (RNase A) is an RNA-cleaving enzyme characterized by its high conformational stability and strong catalytic activity. This enzyme is ubiquitous in living organisms and is difficult to inactivate. In polymerase chain reaction (PCR) RNase activity is removed by adding inhibitors. Molecularly imprinted polymers (MIPs) with high selectivity, high stability, low cost and facile synthesis could prove useful in extraction of target molecules, such as RNase A, from reaction mixtures. In this investigation, MIPs were synthesized from the monomers styrene and polyethyleneglycol 400 dimethacrylate (PEG400DMA) in several different ratios. Styrene as a functional monomer gave MIPs with a higher affinity for RNase A than other functional monomers tested, according to both enzyme-linked immnuosorbent assay (ELISA) and isothermal titration calorimetry (ITC). The optimum volume ratio of styrene/PEG400DMA was 20/100 at 25 degrees C, and this ratio maximized the rebinding efficiency of RNase A to MIPs. Isothermal titration calorimetry was also used, and could be useful to design the composition of molecularly imprinted polymers for various target molecules.

Fabrication of a planar-form screen-printed solid electrolyte modified Ag/AgCl reference electrode for application in a potentiometric biosensor.

This study features the fabrication of a planar-form, solid electrolyte modified, (PSEM) Ag/AgCl reference electrode using a screen-printing method. The PSEM Ag/AgCl reference electrode uses agar gel as the inner electrolyte and chloroprene rubber for the liquid junction and insulator. These common low-cost materials and the simple fabrication processes involved render the proposed reference electrode an ideal candidate for cost-efficient mass production. It is shown that the developed reference electrode is insensitive to most of the physiologically important ionic species, including Na+, K+, Li+, Ca2+, NH4+, and Cl-, under continuous measurement conditions. Moreover, as with conventional commercial reference electrodes, the proposed reference electrode exhibits a reversible response, which is maintained until the agar gel dries out. The PSEM Ag/AgCl reference electrode is integrated with an iridium oxide modified Pt-based pH indicator electrode to form a chip-type pH biosensor. The performance of this biosensor is consistent with that obtained from a pH meter based on a macroscopic commercial Ag/AgCl reference electrode. The experimental results confirm that the proposed biosensor is capable of providing precise pH measurements of various real samples. Accordingly, the PSEM Ag/AgCl reference electrode presented in this study provides a viable alternative to the macroscopic Ag/AgCl reference electrode used in many conventional chip-based pH sensors.

Ionic effect on the binding of bilirubin to the imprinted poly(methacrylic acid-co-ethylene glycol dimethylacrylate).

A molecularly imprinted polymer (MIP) capable of detecting bilirubin was successfully synthesized. Bilirubin template was imprinted in poly(methacrylic acid-co-ethylene glycol dimethylacrylate) [poly(MAA-co-EGDMA)]. MAA and EGDMA were used as the monomer and the cross-linker, respectively. The optimal solvent conditions to maintain its stability were discussed. Solvent system based on ethylenediamine tetraacetic acid (EDTA) and ascorbic acid was compared with respect to the stability of bilirubin. pH and bilirubin concentration were both investigated for the bilirubin stability. Blue light as well as aeration was applied to inspect the regarding effects. The cross-linking effect was further confirmed by the thermogravimetric analysis (TGA). The effect of salts, such as NaCl and KCl on the binding capacity of the molecularly imprinted polymer was also discussed. Further, the rat serum and bile samples were applied and the binding of the MIPs for bilirubin was thus confirmed.

Determination of C-reactive protein with an ultra-sensitivity immunochemiluminometric assay.

C-reactive protein (CRP), the classic acute phase reactant, is strongly associated with increased risk of cardiovascular events. The demand for measuring serum CRP levels has been predicted to increase. We developed an ultra-sensitivity in-house immunometric assay on polystyrene beads for measuring CRP and studied its analytical and clinical performance. The assay used a pair of monoclonal anti-CRP antibodies and detected CRP in a 1-step immunometric assay with a chemiluminescence signal. The calibration was traceable to the World Health Organization reference material. The assay covered a linear range of 0.01 to 50.00 mg/L. The analytical detection limit calculated from the mean level plus 3 SD of the zero calibrator was 0.004 mg/L. The within-run imprecision was 7.0%, 5.2%, and 4.1% for mean CRP levels of 0.02 mg/L, 1.44 mg/L, and 11.04 mg/L, respectively. The between-run imprecision was 9.2%, 7.0%, and 6.0% for mean CRP levels of 0.02 mg/L, 1.49 mg/L, and 10.90 mg/L, respectively. The average recovery was 102.0% (n=6). The assay correlated well with a high-sensitivity latex-enhanced nephelometric assay (regression line y=0.865 x +1.333, r=0.974, S(y/x)=3.415, n=47 for 0-50.00 mg/L and y=1.076 x-0.080, r=0.985, S(y/x)=0.989, n=29 for 0-20.00 mg/L). The central 95 percentile reference interval for Han Chinese residing in Taiwan was 0.02-4.33 mg/L (n=469). There was no significant difference in serum CRP levels between healthy male and female subjects (median, 0.34 and 0.31 mg/L, respectively); however, CRP levels increased moderately with age (r=0.276, P<.05). The reference values for the Chinese population were about 5-fold lower than those for the United States population. This ultra-sensitivity immunochemiluminometric assay for CRP is rapid and accurate and can be used to assess cardiovascular risk.

Microfluidic pH-sensing chips integrated with pneumatic fluid-control devices.

This paper presents a microfluidic chip capable of performing precise continuous pH measurements in an automatic mode. The chip is fabricated using micro-electro-mechanical-systems (MEMS)-based techniques and incorporates polydimethylsiloxane (PDMS) microstructures, pH-sensing electrodes and pneumatic fluid-control devices. Through its enhanced microchannel design and use of pneumatic fluid-control devices, the microfluidic chip reduces the dead volume of the sample and increases the pumping rate. The maximum pumping rate of the developed micro-pump is 28 microL/min at an air pressure of 10 psi and a driving frequency of 10 Hz. The total sample volume consumed in each sensing operation is just 0.515 microL. As a result, the developed chip reduces the sample volume compared to conventional large-scale pH-sensing systems. The microfluidic chip employs the electrochemical sensing method to conduct precise pH level measurements. The sensing electrodes are fabricated by sputtering a layer of SiO(2)-LiO(2)-BaO-TiO(2)-La(2)O(3) (SLBTLO) onto platinum (Pt) electrodes and the pH value of the sample is evaluated by measuring the potential difference between the sensing electrodes and a reference electrode. Additionally, the integration of the microfluidic chip with a pneumatic fluid-control device facilitates automatic sample injection and a continuous sensing operation. The developed system provides a valuable tool with which to examine pH values in a wide range of biomedical and industrial applications.

Synthesis of and recognition by ribonuclease A imprinted polymers.

Ribonuclease (RNase), an enzyme which degrades RNA, is ubiquitous in living organisms, can renature after autoclaving, and is difficult to inactivate. The removal of RNase is especially necessary for the reverse transcription-polymerase chain reaction (RT-PCR) and for in vitro transcription and translation. Typically, RNase inhibitors must be added to these reactions nowadays. Molecularly imprinted polymers (MIPs) could offer many advantages for removal of undesired enzymes, including high binding selectivity, stability, low cost, and facile synthesis. Surface imprinting, employing immobilized RNase, was used in this study to make the most effective use of the template molecules-clearly, inaccessible binding sites, no matter how well imprinted, are not useful for target binding. Different monomers and cross-linkers were used to synthesize RNase-templated MIPs, and the rebinding capacity of each composition was characterized. We found that using polyethylene glycol 400 dimethacrylate (PEG400DMA) gave the highest imprinting effectiveness (i.e. the highest RNase binding ratio between imprinted and non-imprinted polymers). However, including styrene monomer (50 wt%) gave polymers with the highest overall affinity for ribonuclease A (RNase A). Finally, isothermal titration calorimetry was used as an auxiliary tool to help elucidate the mechanisms of the binding of monomers to templates, and ligands to MIPs.