Nitrocellulose/acrylic resin coated screen-printed carbon electrode to construct a capacitive immunosensor for anti-BSA.

The capacitive immunosensor, known for its label-free simplicity, has great potential for point-of-care diagnostics. However, the interaction between insulation and recognition layers on the sensing electrode greatly affects its performance. This study introduces a pioneering dual-layer strategy, implementing a novel combination of acrylic resin (AR) and nitrocellulose (NC) coatings on screen-printed carbon electrodes (SPCEs). This innovative approach not only enhances the dielectric properties of the capacitive sensor but also streamlines the immobilization of recognizing elements. Particularly noteworthy is the superior reliability and insulation offered by the AR coating, surpassing the limitations of traditional self-assembled monolayer (SAM) modifications. This dual-layer methodology establishes a robust foundation for constructing capacitive sensors optimized specifically for liquid medium-based biosensing applications. The NC coating in this study represents a breakthrough in effectively immobilizing BSA, unraveling the capacitive response intricately linked to the quantity of adsorbed recognizing elements. The results underscore the prowess of the proposed immunosensor, showcasing a meticulously defined linear calibration curve for anti-BSA (ranging from 0 to 25 µg/ml). Additionally, specific interactions with anti-HAS and anti-TNF-α further validate the versatility and efficacy of the developed immunosensor. This work presents a streamlined and highly efficient protocol for developing label-free immunosensors for antibody determination and introduces a paradigm shift by utilizing readily available electrodes and sensing systems. The findings are poised to catalyze a significant acceleration in the advancement of biosensor technology, opening new avenues for innovative applications in point-of-care diagnostics.

Self-Healing Hydrogel Containing Decellularized Liver Matrix and Endothelial Cell-Covered Hepatocyte Spheroids for Rescue of Injured Hepatocytes.

Liver fibrosis occurs in many chronic liver diseases, while severe fibrosis can lead to liver failure. A chitosan-phenol based self-healing hydrogel (CP) integrated with decellularized liver matrix (DLM) is proposed in this study as a 3D gel matrix to carry hepatocytes for possible therapy of liver fibrosis. To mimic the physiological liver microenvironment, DLM is extracted from pigs and mixed with CP hydrogel to generate DLM-CP self-healing hydrogel. Hepatocyte spheroids coated with endothelial cells (ECs) are fabricated using a customized method and embedded in the hydrogel. Hepatocytes injured by exposure to CCl4-containing medium are used as the in vitro toxin-mediated liver fibrosis model, where the EC-covered hepatocyte spheroids embedded in the hydrogel are co-cultured with the injured hepatocytes. The urea synthesis of the injured hepatocytes reaches 91% of the normal level after 7 days of co-culture, indicating that the hepatic function of injured hepatocytes is rescued by the hybrid spheroid-laden DLM-CP hydrogel. Moreover, the relative lactate dehydrogenase activity of the injured hepatocytes is decreased 49% by the hybrid spheroid-laden DLM-CP hydrogel after 7 days of co-culture, suggesting reduced damage in the injured hepatocytes. The combination of hepatocyte/EC hybrid spheroids and DLM-CP hydrogel presents a promising therapeutic strategy for hepatic fibrosis.

Determination of o-phthalaldehyde for dose verification of the clinical disinfectant by fluorescent sequential injection analysis.

A new automated, generic analytical approach for determining the clinical disinfectant o-phthalaldehyde (OPA) is reported in this study. The proposed sequential injection analysis (SIA) is based on the online reaction of the OPA with glycine/N-acetylcysteine (NAC) in a neutral medium (pH = 7.0) to form a highly fluorescent isoindole derivative. All critical flow and reaction variables were investigated, while validation was carried out in the linearity detection range (0.0075-0.02%). As a result, excellent linearity (R2 > 0.99) and precision (1.5-2.4% for repeatability and 0.7-2.2% for reproducibility) were achieved for the reference OPA solutions. Furthermore, reasonable concentration verification of OPA disinfection (0.2-0.6%) in healthcare institutes can be achieved using the developed fluorescent SIA due to its good sensitivity (0.111 V/%) and precision (1.0-2.3% for intermediate precision) around the minimum effective concentration (MEC) of 0.3% for Cidex-OPA disinfectant.

An Electrochemical o-Phthalaldehyde Sensor Using a Modified Disposable Screen-Printed Electrode with Polyacrylate Hydrogel for Concentration Verification of Clinical Disinfectant.

The study proposes an o-phthalaldehyde (OPA) sensor for rapid and reliable detection of OPA in healthcare disinfection practices, based on a hydrogel-modified screen-printed carbon electrode strip. The hydrogel film, which contains glycine and N-acetylcysteine, reacts with OPA to produce a reductive isoindole derivative. The derivative is then oxidized for OPA determination using cyclic voltammetry. The proposed sensor achieves an optimal detection time of 20-30 s and requires only a small analyte volume of 5 µL. It exhibits good precision (10%) and sensitivity (3.3 µA/cm2 mM) in a phosphate-buffered solution (pH 7.6), with excellent linearity (R2 > 0.97) and precision (<3%) in the detection range (0.2-0.6%) required for clinical OPA solutions. Moreover, the sensor demonstrates good concentration verification of Cidex-OPA disinfection in healthcare institutes, with high sensitivity (18.28 µA/cm2 mM) and precision around the minimum effective concentration (0.3%). Overall, the proposed sensor offers a promising and practical solution for accurate and reliable OPA detection in clinical disinfection practices.

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.

Determination of hematocrit by voltage-induced hemolysis on a disposable electrochemical sensing strip.

Besides being a crucial parameter for surgery and clinical diagnosis, hematocrit tends to affect the analytical results of point-of-care analytical devices. Therefore, an accurate and quick method for measuring hematocrit was developed on the basis of screen-printed carbon electrodes (SPCE). An impulse DC voltage of 3.0 V was imposed on ferricyanide-coated SPCE to induce hemolysis, and the released hemoglobin reduced the ferricyanide, generating a higher oxidation current for estimating hematocrit. Hematocrit ranging from 10 to 70% can be determined in 5 s by linear sweep voltammetry (r(2) = 0.9907) or 0.8 s by 3 V of potential step voltammetry (r(2) = 0.9833).

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.

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

ß(1→3)-d-Glucan sensors were fabricated using bi-enzyme and tri-enzyme immobilized systems with gold nanoparticles (GNPs) to increase sensitivity. The plant ß(1→3)-D-glucanase (ßG), glucose oxidase (GOD) or/and peroxidase (POD) in agarose-corn flour-gelatin (ACG) matrix were coated on platinum disc electrode to detect soluble ß(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-ßG-GOD-GNPs/Pt) and tri-enzyme sensor (ACG-ßG-GOD-POD-GNPs/Pt) had response time less than 20s for ß(1→3)-D-glucan. A linear calibration plot for bi-enzyme sensor was obtained for ß(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 ß(1→3)-d-glucan content in alcoholic beverages, the results were comparable to those obtained by standard method.

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.

Choline biosensor constructed with chitinous membrane from soldier crab and its application in measuring cholinesterase inhibitory activities.

An amperometric flow-injection choline biosensor was assembled utilizing natural chitinous membrane as the supporting material for biocatalyst immobilization, and the membrane was purified from Taiwanese soldier crab, Mictyris brevidactylus. The chitinous membrane (<50.0 microm in thickness) was covalently immobilized with choline oxidase (EC 3.1.1.17 from Alcaligenes sp.) and then attached onto the platinum electrode of an amperometric flow cell. The flow cell served as the choline sensing device of the proposed FIA system. The sensor signal (peak height of the FIAgram) was linearly related to choline concentration (r=0.999 for choline up to 5.0mM) with low detection limit (S/N>3 for 10.0 microM choline) and high reproducibility (CV<3% for 1.0mM choline, n=7). The system was proved to be useful in measuring cholinesterase inhibitory activities of synthetic chemicals or natural products.

Real-time assay of immobilized tannase with a stopped-flow conductometric device.

A stopped-flow manifold was developed to assay and characterize immobilized tannase (EC 3.1.1.20). The immobilized enzyme reactor was inserted within the tube-type electrode pair (cell constant=103.2 cm(-1)) for a real-time conductometric measurement. Tris buffer (2 mM, pH=7.0) was used as the carrier for sensitivity improvement. The activities and kinetic parameters (Km values) for propyl gallate, methyl gallate and tannic acid were investigated.

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).

Determination of tannin in green tea infusion by flow-injection analysis based on quenching the fluorescence of 3-aminophthalate.

A flow-injection analytical system was developed to determine tannin content in green tea infusions. The flow-injection system is based on measuring the quenching effect of tannin on the fluorescence of 3-aminophthalate. Fluorophore was obtained by auto-oxidation of luminol during solution preparation. System performance was satisfactory for routine analysis (sample throughput >20 h(-1); linear dynamic range for tannic acid, 0.005-0.3 mg/mL; linear dynamic range for green tea tannin, 0.02-1.0 mg/mL; CV < 3%). The flow-injection method is immune from interference by coexisting ascorbate in green tea infusion. Analytical results were verified by the ferrous tartrate method, the Japanese official analytical method.

Formation of tannin-albumin nano-particles at neutral pH as measured by light scattering techniques.

Aggregation phenomena of tannin with bovine serum albumin were investigated by light scattering techniques including photon correlation spectroscopy and Rayleigh scattering. Tannin and albumin formed particles with diameters less than 1 microm at neutral pH. As revealed by this study, light scattering methods are useful in investigating aggregation phenomena of biomolecules and in directly quantifying tannin content.

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 micro A/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 micro M, S/N > 3), reproducible (CV for 55 micro M glucose <3%, n = 5), reagentless, and durable for months.