A Portable Low-Power Acquisition System with a Urease Bioelectrochemical Sensor for Potentiometric Detection of Urea Concentrations.

This paper presents a portable low-power battery-driven bioelectrochemical signal acquisition system for urea detection. The proposed design has several advantages, including high performance, low cost, low-power consumption, and high portability. A LT1789-1 low-supply-voltage instrumentation amplifier (IA) was used to measure and amplify the open-circuit potential (OCP) between the working and reference electrodes. An MSP430 micro-controller was programmed to process and transduce the signals to the custom-developed software by ZigBee RF module in wireless mode and UART in able mode. The immobilized urease sensor was prepared by embedding urease into the polymer (aniline-co-o-phenylenediamine) polymeric matrix and then coating/depositing it onto a MEMS-fabricated Au working electrode. The linear correlation established between the urea concentration and the potentiometric change is in the urea concentrations range of 3.16 × 10(-4) to 3.16 × 10(-2) M with a sensitivity of 31.12 mV/log [M] and a precision of 0.995 (R² = 0.995). This portable device not only detects urea concentrations, but can also operate continuously with a 3.7 V rechargeab-le lithium-ion battery (500 mA·h) for at least four days. Accordingly, its use is feasible and even promising for home-care applications.

Headgroup effects of template monolayers on the adsorption behavior and conformation of glucose oxidase adsorbed at air/liquid interfaces.

Stearic acid (SA) and octadecylamine (ODA) monolayers at the air/liquid interface were used as template layers to adsorb glucose oxidase (GOx) from aqueous solution. The effect of the template monolayers on the adsorption behavior of GOx was studied in terms of the variation of surface pressure, the evolution of surface morphology observed by BAM and AFM, and the conformation of adsorbed GOx. The results show that the presence of a template monolayer can enhance the adsorption rate of GOx; furthermore, ODA has a higher ability, compared to SA, to adsorb GOx, which is attributed to the electrostatic attractive interaction between ODA and GOx. For adsorption performed on a bare surface or on an SA monolayer, the surface pressure approaches an equilibrium value (ca. 8 mN/m) after 2 to 3 h of adsorption and remains nearly constant in the following adsorption process. For the adsorption on an ODA monolayer, the surface pressure will increase further 1 to 2 h after approaching the first equilibrium pressure, which is termed the second adsorption stage. The measurement of circular dichroism (CD) spectroscopy indicates that the Langmuir-Blodgett films of adsorbed GOx transferred at the first equilibrium state (π = 8 mN/m) have mainly a ß-sheet conformation, which is independent of the type of template monolayers. However, the ODA/GOx LB film transferred at the second adsorption stage has mainly an α-helix conformation. It is concluded that the specific interaction between ODA and GOx not only leads to a higher adsorption rate and adsorbed amount of GOx but also induces a conformation change in adsorbed GOx from ß-sheet to α-helix. The present results indicate that is possible to control the conformation of adsorbed protein by selecting the appropriate template monolayer.

A multi-functional electrochemical sensing system using microfluidic technology for the detection of urea and creatinine.

This study presents a new microfluidic system capable of precise measurements of two important biomarkers, urea and creatinine, automatically. In clinical applications, high levels of these two biomarkers are early indicators of nephropathy or renal failure and should be monitored on a regular basis. The microfluidic system is composed of a microfluidic chip, a control circuit system, a compressed air source and several electromagnetic valves to form a handheld system. The microfluidic chip is fabricated by using micro-electromechanical systems and microfluidic techniques comprising electrochemical sensor arrays and polydimethylsiloxane-based microfluidic structures such as micropumps/micromixers, normally closed valves and microchannels. The microfluidic system performs a variety of critical processes including sample pretreatment, mixing, transportation and detection on a single chip. The experimental results show that the entire procedure takes approximately 40 min, which is much faster than the traditional method (more than 6 h). Furthermore, the total sample volume consumed in each operation is only 0.1 mL, which is significantly less than that required in a large system (5 mL). The developed automatic microfluidic system may provide a powerful platform for further clinical applications.

Synthesis of recognition matrix from 4-methylamino-N-allylnaphthal-imide with fluorescent effect for the imprinting of creatinine.

4-Methylamino-N-allylnaphthalimide (4-MAANI), a functional monomer with fluorescent effect, was synthesized for the imprinting and specific uptake of creatinine, an important clinical marker for kidney function. 4-Methylamino-N-allylnaphthalimide was synthesized from the reaction of 4-bromo-1,8-naphthalic anhydride with allylamine to form 4-bromo-N-allylnaphthalimide and further to react with methylamine. Excitation and emission of the fluorescent monomer was investigated by both a three-dimensional plot of fluorescent intensity versus excited and emitted wavelengths and a corresponding contour plot. The photoluminescence properties of the as-prepared conjugated species were also studied. 4-Methylamino-N-allylnaphthalimide (4-MAANI) fluorescent monomer, methacrylic acid (MAA) functional monomer, ethylene glycol dimethylacrylate (EGDMA) cross-linker, and 2,2'-azobisisobutyronitrile (AIBN) initiator were then utilized in the presence of creatinine template for the processing of heat-induced polymerization. FT-IR analysis was used to confirm the successful synthesis of 4-MAANI. The specific recognition cavity for creatinine was then created from the polymer matrix after the removal of creatinine by proper solvent. The imprinting effect as well as selectivity from the creatinine containing mixture was also evaluated. The grouped clusters from the emitted fluorescent intensities of the imprinted and nonimprinted polymers before and after rebinding of creatinine were obtained and discussed. Serum spiked with a different concentration of creatinine was also used to confirm the feasibility of clinical applications in the future. Successful recognition of the creatinine molecule via the combined effect of molecular imprinting and photoluminescence of the imprinted polymeric material was thus confirmed in this work.

Ionic effect investigation of a potentiometric sensor for urea and surface morphology observation of entrapped urease/polypyrrole matrix.

Potentio-dynamic polymerization of buffered urease and pyrrole monomer onto carbon papers was conducted to fabricate an immobilized urease electrode for measuring the urea concentration. To use carbon paper as the substrate for the electro-growth of polypyrrole matrix not only created sufficient adhesion of the conducting polymer layer but also provided superior entrapment of urease enzymes. The potentiometric response corresponding to ammonia, the product formed from the urease catalyzed urea reaction, was employed for the urea concentration measurement. Scanning electron microscopic photographs showed that the polypyrrole matrix deposited on the carbon papers appeared to be of a cylindrical nanotube shape. The charge density applied in the polymerization was found to affect the potentiometric response while the potential-scanning rate showed minor influence. The composite electrodes had high sensitivity in urea detection, showing a response linear to the logarithm of the urea concentration in the range of 10(-3) to 10 mM. The detection of urea solution prepared in water and buffer was also compared. Ionic effect on the sensing of urea solution was investigated. By comparing the data reported in literature, the urease/polypyrrole/carbon paper electrode developed in this work showed superior long-term stability and reusability. The detection of urea in serum was also well performed.

Synthesis of an imprinted hybrid organic-inorganic polymeric sol-gel matrix toward the specific binding and isotherm kinetics investigation of creatinine.

Hybrid organic-inorganic polymeric sol-gel materials imprinted with creatinine template molecules were synthesized for the specific binding of creatinine. Creatinine is a metabolite from creatine and is the final product from kidney metabolism. Therefore, creatinine can be an important index to estimate the function of the kidney. It was then chosen as the target molecule in this work. To achieve the specific binding toward creatinine, molecular imprinting was used to create a polymeric matrix for the regarding purpose. Sol-gel was further added to create a rigid network structure for the absorption of creatinine. An inorganic precursor, tetraethoxysilane (TEOS), was mixed with an organic functional monomer, 2-acrylamido-2-methylpropane-sulfonic acid (AMPS), and the creatinine template to form a hybrid organic-inorganic imprinted polymer. The chemical functionality was achieved as well as a confined matrix via the polymerization and the hydrolysis-condensation of the sol-gel. The imprinting effect from the hybrid materials against the corresponding nonimprinted was investigated. BET (Brunauer-Emmett-Teller) analysis was carried out for the imprinted and the nonimprinted materials. The specificity of the hybrid materials was further examined by capping the surface silanol groups with chloro-trimethylsilane (CTMS) and 1,1,1,3,3,3-hexamethyldisilazane (HMDS), respectively. The capping effect was compared and discussed from the binding results. Selectivity of the materials toward creatinine was obtained using mixture solutions in the presence of creatinine and its analogues. Reutilization and storage stability of the hybrid organic-inorganic imprinted material were also studied. Additionally, the affinity distribution of the hybrid imprinted materials derived from the allosteric model was also analyzed from the adsorption isotherm data.

Via zinc(II) protoporphyrin to the synthesis of poly(ZnPP-MAA-EGDMA) for the imprinting and selective binding of bilirubin.

Poly(zinc protoporphyrin-methacrylic acid-ethyl glycol dimethylacrylate) (poly(ZnPP-MAA-EGDMA)) imprinted with alpha-bilirubin can cause spectroscopic change in wavelength and absorption intensity due to the metal-ion coordination between ZnPP and bilirubin. The fluorescent imprinted polymer was able to selectively bind alpha-bilirubin. The corresponding imprinted polymer monolith was synthesized by using the functional monomer, methacrylic acid and the fluorescent monomer, zinc(II) protoporphyrin. Although the imprinted polymers (MIPs) using methacrylic acid, protoporphyrin, or zinc(II) protoporphyrin alone as the only functional monomer could bind bilirubin, the imprinting effects were all comparably inferior to the imprinted poly(ZnPP-MAA-EGDMA). Therefore, it revealed that via the combined utilization of ZnPP and MAA for the fluorescent and functional effect, the MIPs thus prepared were then able to create the highly selective cavities. The optimal condition for the heated polymerization of the imprinted poly(ZnPP-MAA-EGDMA) was found to be 60 degrees C for 6 h. The imprinting factor of 3.069 could be achieved from the fluorescent imprinted polymer by comparing the binding results obtained from the MIP and the NIP (non-imprinted polymer). The imprinting factor obtained from bilirubin/biliverdin mixture solution was reduced to 2.111 because of the presence of biliverdin. The selectivity toward bilirubin of 2.269 from the bilirubin/biliverdin mixture was obtained. Therefore, to utilize ZnPP for the preparation of the imprinted materials confirmed the selective binding and detection of bilirubin via the fluorescent approach.

Diagnostic role of biliary pancreatic elastase for cholangiocarcinoma in patients with cholestasis.

BACKGROUND: A wide array of proteins is secreted into the bile and may be associated with biliary tract diseases. We attempted to discover novel biomarker in bile for cholangiocarcinoma. METHODS: Bile was collected from patients with bile duct obstruction. Proteins were separated by 2-dimensional electrophoresis and identified by mass spectrometry. Levels of mRNA and protein expression of the candidate biomarker were analyzed by real-time PCR and Western blotting, respectively, whereas enzyme activity was measured by a kinetic method. The diagnostic efficacy was assessed by receiver operating characteristic (ROC) curve analysis. RESULTS: Pancreatic elastase (PE) 3B was identified as a biomarker for cholangiocarcinoma. The mRNA of PE 3B was up-regulated in cancerous tissues, compared to non-cancerous tissues. The protein expression and enzyme activity of PE in bile were increased in patients with cholangiocarcinoma, compared to gallstone patients. Biliary amylase activity was used to correct the presence of pancreaticobiliary reflux. Significantly higher PE/amylase ratios in bile were found in patients with cholangiocarcinoma (0.214+/-0.045) than those with gallstone (0.023+/-0.005, p<0.001). The area under the ROC curve of the ratio was 0.877 (95% CI: 0.765 to 0.988). Using 0.065 as a cutoff value, the ratio distinguished malignant from benign causes of biliary obstruction with a sensitivity of 82% and a specificity of 89%. CONCLUSION: PE in bile is a biomarker for cholangiocarcinoma and the combination measurement of PE and amylase enhances diagnostic efficacy.

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.

Amperometric detection of bilirubin from a micro-sensing electrode with a synthetic bilirubin imprinted poly(MAA-co-EGDMA) film.

Poly(methacrylic acid-co-ethyl glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was shown to be able to bind alpha-bilirubin in our previous work. In this work, the corresponding imprinted polymer thin film was synthesized onto a thiol treated Au electrode by surface grafting polymerization. Bilirubin was able to be detected by an Au electrode, however, the electrode was not be able to discriminate bilirubin from the other matrix components if clinical samples were used. Therefore, the imprinted material was introduced so that the modified Au electrode could specifically detect bilirubin. Optimal potential was found to be 0.55 V and this was set for the rest of experiments. The imprinting factor of 3.16 was confirmed by comparing the signals from the MIP-Au and the NIP (non-imprinted polymer)-Au electrode. Calibration of the bilirubin concentration with respect to the current by the MIP-Au electrode was made within the range of 5mg/dl and a detection sensitivity of 0.644 microA/mg/dl (2.58 microA/cm(2)/mg/dl) was obtained. Furthermore, a linear correlation of the bilirubin concentration within 1.0mg/dl versus detection current was also achieved. Bilirubin was further detected by the MIP-Au electrode in the presence of fetal bovine serum (FBS). Repeated detection of bilirubin with at least three detection batches was performed and the reproducibility of the same piece of MIP-Au electrode was confirmed. The result was compared to those obtained from the serum and the solvent solution. The results indicated the feasibility of using the bilirubin imprinted poly(MAA-co-EGDMA) film as a sensing electrode for the clinical detection of bilirubin in serum.

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.

Synthesis of bilirubin imprinted polymer thin film for the continuous detection of bilirubin in an MIP/QCM/FIA system.

A bilirubin imprinted polymer (BIP) was coated on a thiol pretreated Au electrode on a quartz crystal microbalance (QCM) chip. The BIP thin film was synthesized using 4-vinylpyridine (4-Vpy) as the monomer, divinylbenzene (DVB) as the cross-linker, and benzophenone as the initiator. By using a photo-graft surface polymerization technique with irradiation by ultra-violet (UV) light, a thin BIP film was prepared, from which a biomimetic sensor for the detection of bilirubin was developed. The sensor was able to discriminate bilirubin in solution owing to the specific binding of the imprinted sites. The BIP/QCM chip has been repeatedly used for more than 7 months in many continuous experiments. The detection signal of bilirubin from the BIP thin film/QCM was compared with the non-BIP thin film/QCM. Biliverdin, an analogue of bilirubin, was used for comparison. The analogue comparison confirmed the binding specificity of the BIP film toward bilirubin. The selectivity can be as high as 31.2. The effect of pH on the detection of bilirubin is also discussed. With proper solvent for elution and recovery, flow injection analysis (FIA) could be applied to the system. The performance of the BIP/QCM chip was evaluated. A linear calibration of the bilirubin concentration with respect to the frequency shift was successfully obtained. The reproducibility of measurements from the same BIP/QCM chip was confirmed. In addition, repeatability of detection was also confirmed from different BIP/QCM chips. In conclusion, a combined BIP thin film/QCM/FIA method was successfully established for the detection of bilirubin concentration using a molecularly imprinted film.

Designing a molecularly imprinted polymer as an artificial receptor for the specific recognition of creatinine in serums.

In this study, molecularly imprinted polymers (MIP) synthesized from two different functional monomers, beta-cyclodextrin (beta-CD) and 4-vinylpyridine (4-Vpy), were prepared. The crosslinkers used for these two monomers were epichlorohydrin (EPI) and divinylbenzene (DVB), respectively. It was attempted to adsorb the target molecule, creatinine, from its mixture solutions. A proper molar ratio of monomer/crosslinker for the preparation of the imprinted poly(beta-CD) was 1:10. Between both polymers mentioned above, the affinity of the imprinted poly(4-Vpy-co-DVB) towards creatinine was comparably superior. The imprinted poly(4-Vpy-co-DVB) for creatinine could reach a specific binding ratio of 3.11. The imprinted poly(4-Vpy-co-DVB) was further utilized to bind creatinine from human serum samples. The binding capacity of the imprinted poly(4-Vpy-co-DVB) for creatinine from the serum samples was plotted against the creatinine concentration. From the correlation, the feasibility of the imprinted poly(4-Vpy-co-DVB) thus prepared for the target analyte, creatinine, was experimentally confirmed.

Development and validation of an HPLC-UV method for the analysis of methoxyamine using 4-(diethylamino)benzaldehyde as a derivatizing agent.

Methoxyamine (MX) is a potential new anti-cancer drug. In this paper, a quantitative HPLC-UV method for MX using 4-(diethylamino)benzaldehyde (DEAB) as a derivatizing agent has been developed and validated. The studies showed that MX reacts with DEAB under acidic conditions to form protonated 4-(diethylamino)benzaldehyde o-methyloxime (DBMOH+). The equilibrium between DBMOH+ and its conjugate base 4-(diethylamino)benzaldehyde o-methyloxime (DBMO) is affected by both buffer concentration and organic solvent content in the solution. The method developed uses a reversed phase C18 column for the separation of MX derivatives, an internal standard benzil for method calibration, and a UV detector at a wavelength of 310 nm for analyte detection. The MX derivatives can be resolved in ca. 20 min. The method has a linear calibration range from 0.100 to 10.0 microM with a correlation coefficient of 0.999 for MX and a detection limit of 5 pmol with a 50 microl sample size. The intra-assay and inter-assay precision expressed in terms of percent relative standard deviation were < or =5 and 8%; and the intra-assay and inter-assay accuracy defined as the measured value divided by the accepted value multiplied by 100% were 94.2-100 and 92.6-111%, respectively. This method may be used for the analysis of MX in pharmaceutical preparations.

Binding specificity of alpha-bilirubin-imprinted poly(methacrylic acid-co-ethylene glycol dimethylacrylate) toward alpha-bilirubin.

alpha-Bilirubin is an important index to determine the liver's functions. Poly(methacrylic acid-co-ethylene glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was proposed and shown to be able to bind alpha-bilirubin specifically. The extraction condition was also discussed. Polymers prepared by imprinting bilirubin in poly(MAA-co-EGDMA) and in poly(beta-cyclodextrin-co-EGDMA) were compared. In this work, binding specificity of molecularly imprinted polymer (MIP) toward the target template, alpha-bilirubin, is discussed. Adsorption profile of alpha-bilirubin by bilirubin-imprinted poly(MAA-co-EGDMA) was measured as a function of time, from where the equilibrium could be determined. Two hours was determined to be the proper time for adsorption. Biliverdin as a rather similar analog compound of bilirubin was chosen for comparison of binding specificity in this study. Progesterone as well as testosterone was also chosen for study because they both co-exist in serum with bilirubin and might interfere with binding capacity of imprinted poly(MAA-co-EGDMA) toward alpha-bilirubin. Specificity of this polymer for bilirubin was thus confirmed by tasks carried out in mixture solutions comprised of compounds above. It is true that biliverdin contained in the binary mixture did affect the binding capacity of bilirubin. Nevertheless, polymer's binding specificity for bilirubin was essentially sufficient for recognition of alpha-bilirubin in the presence of other compounds. How MIP identified the target template molecule, alpha-bilirubin, is also elucidated.

Synthesis of creatinine-imprinted poly(beta-cyclodextrin) for the specific binding of creatinine.

An artificial receptor for creatinine was synthesized by the method of molecularly imprinted polymer (MIP). beta-Cyclodextrin was used as a monomer cross-linked with epichlorohydrin in the presence of creatinine, which was a template for the imprinting. Different molar ratios of monomer to template were used to synthesize the polymers so that better specific adsorption ability towards creatinine could be achieved. The results showed that to carry out the polymerization with a molar ratio of monomer to template of 3:2 and monomer to cross-linking agent of 1:10 was proper. N-hydroxysuccinimide and 2-pyrrolinidone were used as the analogues of creatinine in the adsorption experiments of multi-component solutions to reveal the specific recognition ability of the molecularly imprinted poly(beta-cyclodextrin) (poly(beta-CD)) for the template molecule, creatinine. One such detection interference of creatinine was creatine, which also co-exists in serum. Hence, adsorption experiments of creatinine/creatine binary mixture were also carried out to investigate and confirm the specific binding of the creatinine-imprinted poly(beta-cyclodextrin) towards creatinine. The hydroxyl groups of the imprinted poly(beta-CD) was further capped by chlorotrimethylsilane (CTMS) to investigate the interaction between creatinine and the imprinted poly(beta-CD). The adsorption resulting from the mixture solution by MIPs suggested that the creatinine-imprinted poly(beta-CD) demonstrated superior binding effect for the target molecule, creatinine, rather than creatine, N-hydroxysuccinimide and 2-pyrrolinidone.

Modeling of immobilized cell columns for bioconversion and wastewater treatment.

Immobilized cells are widely used in bioconversions to produce biological products as well as in wastewater treatment such as solvent removal from wastewater streams. In this work, a rate model is proposed to simulate this kind of process in an axial-flow fixed-bed column packed with porous particles containing immobilized cells. The transient model considered various mass transfer mechanisms including axial dispersion, interfacial film mass transfer, and intraparticle diffusion. Cell death in the immobilized cell system was also considered. Effects of various parameters such as kinetic constants and mass transfer parameters were studied. Operational situations such as feed fluctuation flow rate increase and two columns in series were also investigated. The model can be used to study the behavior and characteristics of immobilized cell columns in order to perform scale-up predictions of effluent profiles and for the purpose of process optimization.