Hand-held Colorimetry Sensor Platform for Determining Salivary α-Amylase Activity and Its Applications for Stress Assessment.

This study develops a hand-held stress assessment meter with a chemically colorimetric strip for determining salivary α-amylase activity, using a 3,5 dinitrosalicylic acid (DNS) assay to quantify the reducing sugar released from soluble starch via α-amylase hydrolysis. The colorimetric reaction is produced by heating the strip with a mini polyester heater plate at boiling temperature to form a brick red colored product, which measured at 525 nm wavelength. This investigation describes in detail the design, construction, and performance evaluation of a hand-held α-amylase activity colorimeter with a light emitted diode (LED) and photo-detector with built-in filters. The dimensions and mass of the proposed prototype are only 120 × 60 × 60 mm³ and 200 g, respectively. This prototype has an excellent correlation coefficient (>0.995), comparable with a commercial ultraviolet⁻visible spectroscope, and has a measurable α-amylase activity range of 0.1⁻1.0 U mL-1. The hand-held device can measure the salivary α-amylase activity with only 5 µL of saliva within 12 min of testing. This sensor platform effectively demonstrates that the level of salivary α-amylase activity increases more significantly than serum cortisol, the other physiological stressor biomarker, under physiologically stressful exercise conditions. Thus, this work demonstrates that the hand-held α-amylase activity meter is an easy to use and cost-effective stress assessment tool for psychoneuroendocrinology research.

Label-free and reagentless capacitive aptasensor for thrombin.

This investigation develops a label-free and reagentless aptasensor, based on a capacitive transducer with simple face-to-face electrode pairs. The electrode pairs of the transducer are composed of a gold electrode and an indium tin oxide film with micrometer separation with a double-side polyethylene terephthalate tape. Aptamers and 1-dodecanethiol are modified to form a self-assembled monolayer (SAM) on the gold electrode surfaces, and function as bio-recognition elements and preventers of non-specific protein binding, respectively. Electrochemical characterization results indicate that the SAM also forms an effective insulating layer, which is sufficient for capacitive sensing. The feasibility of the capacitive biosensor is validated using thrombin as a model analyte. The ultra-small value changes of capacitance originating from thrombin binding with the aptamers modified on the biosensor were measured with a home-made capacitance measuring circuit based on switched capacitor (SC) technology. The developed biosensor has detection limits of 1 pM and 10 pM of thrombin in phosphate buffered saline and mimic serum solution, respectively. The linear range for thrombin detection in human serum solution is from 10 pM to 1 µM, with a regression coefficient of 0.98. Additionally, the proposed aptasensor does not have significant levels of non-specific binding of bovine serum albumin and human serum albumin. Accordingly, the combination of SC and SAM bringing capacitive transduction at the forefront of ultrasensitive label-free and reagentless biosensing devices, particularly for point-of-care clinical analysis, which adopts small numbers of biological samples with low analyte concentrations.

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.

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.

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).

Determination of camptothecins in DMSO extracts of Nothapodytes foetida by direct injection capillary electrophoresis.

A rapid capillary electrophoresis procedure was developed for determining the anti-cancer components, camptothecins, in Nothapodytes foetida. The hydrophobic compound was extracted from plant tissue (ca. 1 mL of DMSO for 100 mg of dried plant tissue) with a water-miscible organic solvent, DMSO, at elevated temperature (60 degrees C). The extract was directly injected into the separation capillary (untreated fused silica, 34 cm in length, 75 microm i.d.) and analysed in MEKC mode (369 nm). Within 5 min of migration, camptothecins were successfully separated and quantified by adding organic modifiers to the running buffer (20% DMSO, 90 mm SDS in 10 mm borate buffer, pH 8.60). The linear dynamic range for camptothecin was from 5 to 400 microg/mL. This method was proven to be very suitable for monitoring the amount of camptothecins during the cultivation of the medicinal plant.

Characterization of natural chitosan membranes from the carapace of the soldier crab Mictyris brevidactylus and its application to immobilize glucose oxidase in amperometric flow-injection biosensing system.

This study investigated characteristics of a chitosan membrane from the carapace of the soldier crab Mictyris brevidactylus intended to construct an amperometric biosensor. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used in this study to characterize these chitosan membranes intended for constructing enzymatic biosensors. Chitosan membranes suffering various durations (>10 min) of deacetylation had small charge-transfer resistances (<7.88 kohms) but large double-layer capacitances (>0.55 microF). They were found in EIS where both the solution resistance and Warburg impedance upon electrode interface were almost independent of the durations and degree of deacetylation. The degree of deacetylation and the thickness of chitosan membranes were also determined. Membrane thickness was slightly dependent with the duration but degree of deacetylation was slightly dependent on the duration. Chitosan membranes with various thicknesses suffered various durations of deacetylation, but this did not influence their electrochemical characteristics. The chitinous membrane was covalently immobilized with glucose oxidase (EC 1.3.4.3) and then attached onto the platinum electrode of a homemade amperometric flow cell. Sensor signal was linearly related to glucose concentration (r=0.999 for glucose up to 1.0 mM). The system was sensitive (S/N>5 for 10 microM glucose) and reproducible (CV<1.3% for 50 microM glucose, n=5).

Spectrophotometric determination of deacetylation degree of chitinous materials dissolved in phosphoric acid.

A simple spectrophotometric method is proposed for determining deacetylation degrees (DD) of chitinous materials using phosphoric acid as the UV-transparent solvent system. Calibrating by the extinction coefficients (A(210)) of D-glucosamine and N-acetyl-D-glucosamine, DD values (24-88%) were computed numerically. The results correlated well (R(2) = 0.9805, n = 50) with those obtained by solid-state (13)C NMR. Comparison of the results obtained by the proposed UV method and solid-state (13)C NMR.