Richie L.C. Chen

Sequential enzymatic monitoring of glucose, ethanol and glutamate in bioreactor fermentation broth containing a high salt concentration by a multi-channel flow-injection analysis method

An automated multi-channel flow-injection analysis (FIA) system was developed and its application as an on-line multi-component sensing device for bioprocess monitoring was demonstrated. For system development, an automated glucose analyzer was modified by installing a column switching valve downstream from the injector, and into each channel an immobilized enzyme (oxidase) mini-reactor was inserted. The product (hydrogen peroxide) from each enzymatic reaction was detected electrochemically. Several components (up to six) can be measured sequentially or in a programmed sequence by a computer controlling the electrical valve described above. To test the utility of the present system, the following fermentation process was utilized. Aged fish sauce was transferred into a fermenter loaded with Torulopsis versatiles immobilized beads, and the fermentation process was triggered by the addition of glucose. During the whole process, the glucose level was monitored periodically and the contents of ethanol and glutamate were measured when the glucose content dropped to a certain level. Since the unusually high salt content (17.9%) in the fermentation broth affected the measurement, an additional mini-reactor packed with an ion-exchange resin was inserted between the injector and the column switching valve to stabilize the baseline. As another novel aspect, this anion-exchange column can be used without regeneration, which is valuable for long-term use.

A hand-held electronic tongue based on fluorometry for taste assessment of tea.

A hand-held electronic tongue was developed for determining taste levels of astringency and umami in tea infusions. The sensing principles are based on quenching the fluorescence of 3-aminophthalate by tannin, and the fluorogenic reaction of o-phthalaldehyde (OPA) with amino acids to determine astringency and umami levels, respectively. Both reactions were measured by a single fluorescence sensing system with same excitation and emission wavelengths (340/425 nm). This work describes in detail the design, fabrication, and performance evaluation of a hand-held fluorometer with an ultra-violet light emitted diode (UVLED) and a photo-detector with a filter built-in. The dimension and the weight of proposed electronic tongue prototype are only 120×60×65 mm(3) and 150 g, respectively. The detection limits of this prototype for theanine and tannic acid were 0.2 μg/ml and 1 μg/ml, respectively. Correlation coefficients of this prototype compared with a commercial fluorescence instrument are both higher than 0.995 in determinations of tannin acid and theanine. Linear detection ranges of the hand-held fluorometer for tannic acid and theanine are 1-20 μg/ml and 0.2-10 μg/ml (CV<5%, n=3), respectively. A specified taste indicator for tea, defined as ratio of umami to astringency, was adopted here to effectively distinguish flavour quality of partially fermented Oolong teas.

Electrochemical β(1→3)-d-glucan biosensors fabricated by immobilization of enzymes with gold nanoparticles on platinum electrode.

β(1→3)-d-Glucan sensors were fabricated using bi-enzyme and tri-enzyme immobilized systems with gold nanoparticles (GNPs) to increase sensitivity. The plant β(1→3)-D-glucanase (βG), glucose oxidase (GOD) or/and peroxidase (POD) in agarose-corn flour-gelatin (ACG) matrix were coated on platinum disc electrode to detect soluble β(1→3)-D-glucan. The atomic force microscopy (AFM) revealed that GNPs embedded in ACG formed tiny islands/clusters with enzymes. Both of bi-enzyme sensor (ACG-βG-GOD-GNPs/Pt) and tri-enzyme sensor (ACG-βG-GOD-POD-GNPs/Pt) had response time less than 20s for β(1→3)-D-glucan. A linear calibration plot for bi-enzyme sensor was obtained for β(1→3)-D-glucan concentration ranged from 100 to 1000 ngmL(-1) (R(2)=0.983). The lower detection limit was 30 ngmL(-1) using applied potential of 200 mV and scan rate of 50 mVs(-1); with signal to noise ratio (S/N) of 3. Fabricated tri-enzyme sensor was also operable under similar conditions with LOD of 50 ngmL(-1) (r(2)=0.989) at -175 mV applied potential and scan rate of 50 mVs(-1). Both sensors were durable and could be repeatedly used for at least 14 times. When the tri-enzyme sensor was employed to analyze β(1→3)-d-glucan content in alcoholic beverages, the results were comparable to those obtained by standard method.

Assays for serum cholinesterase activity by capillary electrophoresis and an amperometric flow injection choline biosensor

A capillary electrophoresis method and a durable choline biosensor were developed for measuring serum cholinesterase (EC 3.1.1.8) activity, a useful clinical index for liver function. The former is based on separation of benzoate and benzoylcholine (the artificial substrate of cholinesterase) in an uncoated fused-silica capillary. The migration time of benzoylcholine and benzoate was 1.3 min and 5.5 min, respectively. By the peak areas of A(233) signals, the linear dynamic ranges for both analytes were 0.01-50.0 mM, and the relative standard deviations of 1.0 mM benzoylcholine and benzoate were less than 4% and 6%, respectively. The FIA-choline sensor was constructed with the working electrode of the flow cell covered with a natural chitinous membrane purified from Taiwanese soldier crab, Mictyris brevidactylus. The biomembrane served as the supporting material for enzyme immobilization (choline oxidase, EC 1.1.3.17), and also prevented protein adsorption on the electrode surface. The calibration curve was linear between 0.05 and 5.0 mM (r=0.999). The relative standard deviations for 1.0 mM choline (n=7) were less than 3%, and the activity of the bioactive membrane lasted for about 2 months. The analytical results of both methods correlated well (r=0.940).

High-Speed Mass Measurement of Nanoparticle and Virus

Until now, there have been no relatively easy methods to measure the mass and mass distributions of nanoparticles/viruses. In this work, we report the first set of measurements of mass and mass distributions for nanoparticles/viruses using a novel mass spectrometry technology. In the past, mass spectrometry was typically used to measure the mass of a particle or molecule with a mass less than 1,000,000 Da. We developed cell mass spectrometry that can measure the mass of a cell or a microparticle. Nevertheless, there is a gap for mass measurement methods in the mass region of a nanoparticle or virus (1 MDa to 1 GDa). Here, we developed a nanoparticle/virus mass spectrometry technique to make rapid and accurate mass and mass distribution measurements of nanoparticles/viruses. This technique should be valuable for the quality control of nanoparticle production and the identification of various viruses. In the future, this method can also serve to monitor drug delivery when nanoparticles are used as carriers. Furthermore, it may be possible to measure the degree of infection by measuring the number of viruses in specific cells or in plasma.

Use of Chitosan Membrane from the Carapace of the Soldier Crab Mictyris brevidactylus for Biosensor Construction

Glucose oxidase (EC 1.3.4.3) was immobilized on chitosan membrane (<0.1 mm in thickness) prepared from the carapace of the soldier crab Mictyris brevidactylus. A glucose electrode was constructed by covering a platinum electrode (2.0 mm in diameter) with the enzyme membrane. The enzyme electrode sensed glucose amperometrically (1.0 µA/mM, with linear range up to 0.5 mM, r = 0.999) when positively imposed with 0.6 V against an Ag/AgCl reference electrode. The glucose biosensor was sensitive (<0.1 µM, S/N > 3), reproducible (CV for 55 µM glucose <3%, n = 5), reagentless, and durable for months.

Quantification of 5‐aminolevulinic acid by CE using dynamic pH junction technique

An online dynamic pH junction preconcentration method was developed for quantification of 5-aminolevulinic acid (ALA) by CE with the separation time less than 6 min. The optimal dynamic pH junction of ALA was carried out between pH 9.3 borate buffer (BGE, 40 mM) and pH 2.5 phosphate buffer (sample matrix, 40 mM) when 4.1 cm of sample plug was hydrodynamically injected into an uncoated fused-silica capillary (48.5 cm in length, id of 50 μm). If a 24 kV separation voltage was applied, the calibration curve of ALA peak area (200 nm) showed good linearity (R(2) = 0.9991) ranging from 0.01 to 6.5 mg/mL. The reproducibility of this system was excellent with RSDs (n = 10) of 2.5% for peak area response and 0.6% for migration time at ALA concentration of 0.5 mg/mL. The LOD was evaluated as 1.0 μg/mL (S/N > 3). Compared to conventional CE procedure, the sensitivity was successfully improved over 50-fold. The analytical results of pharmaceutical formulations show a good agreement with those by HPLC (r = 0.94).

Impedimetric sensing of the ethanol and water contents in gasohol with a flow-through carbon electrode pair

The ethanol in gasohol was estimated with a flow-through hydrophobic electrode pair (two identical glassy carbon electrodes, cell constant = 0.3 cm−1). Based on the impedimetric signal at 320 Hz, the important fuel quality parameter can be measured in less than 1 s with sufficient temperature tolerance (20–40 °C) and reproducibility (relative standard deviation ≤ 2% for ten tests). The water content can also be estimated by comparing the impedance data obtained with and without desiccation.

Judgment of pure fermented soy sauce by fluorescence resonance energy transfer of OPA-tryptophan adduct

Tryptophan was detected with a flow-injection manifold after reacting with mM order of fluorogenic o-phthalaldehyde (OPA)/thiol reagent (pH 10.0) in the carrier stream (0.63 mL/min). Based on the intra-molecular fluorescence resonance energy transfer of OPA-tryptophan adduct, the difference in fluorescence intensity obtained at 280 and 300 nm excitation was used to detect tryptophan content with satisfactory precision (CV<6.5% for concentration higher than 0.5 μM), linearity (0.1-10 μM, R(2)=0.9893) and sensitivity (≈10 nM). Since tryptophan will decompose during manufacturing non-fermented soy sauce by acid-hydrolysis procedure, the method was used to discriminate pure fermented soy sauces, adulterated soy sauces and chemical soy sauces in less than 5 min. The ratio of tryptophan to total amino acid content served as the index for the judgment, and the results were validated by capillary electrophoresis.

Single-scan measurement of conductance of a quartz crystal microbalance array coupled with resonant markers for biosensing in liquid phase

This work presents a method for sensing the viscoelastic property of liquid/solid interface using a quartz crystal microbalance (QCM) array. Each sensor in a QCM array has a unique resonant frequency and can be identified by a single-scan measurement of admittance (or impedance). The resonant frequency encoding at each sensor in an array was realized by connecting a capacitor with a known capacitance, called a resonant marker, to the sensor in series. Changes in the resonant frequency of all sensors in an array can be determined using an impedance analyzer and a program that determines the frequencies at which the conductance is at a local maximum. The sensing method allows every sensor output (resonant frequency) to be obtained without the use of time-consuming multiplexed hardware and software. Adsorptions of biomolecules by multiple sensor are monitored in the liquid phase to demonstrate the feasibility of frequency encoding using resonant markers and the single-scan measurement of conductance of a QCM array.