Simple colorimetric assay using pectin hydrogel reagent coupled with camera-based photometry for trace arsenic determination.

Humans mainly ingest arsenic through contaminated drinking water, causing serious health effects. The World Health Organization (WHO) has set the permissible limit of arsenic in drinking water at 0.01 mg/L and concentrations should be regularly determined to ensure a safe supply. In this study, a leucomalachite green (LMG) pectin-based hydrogel reagent was prepared that selectively reacted with arsenic over other metals including manganese, copper, lead, iron, and cadmium. Pectin, optimized at 0.2% (w/v), was used to form the hydrogel matrix. Arsenic reacts with potassium iodate in sodium acetate buffer medium to liberate iodine that then oxidizes LMG entrapped in pectin hydrogel to form a blue product. Camera-based photometry/ImageJ software was used to monitor the color intensity, eliminating the need for a spectrophotometer. The intensity of gray in the red channel was chosen as optimal for the red, green, and blue (RGB) analysis. The colorimetric assay revealed a dynamic detection range toward arsenic solution standards of 0.003-1 mg/L, covering the WHO recommendation of below 0.01 mg/L arsenic in drinking water. The assay gave recovery rates between 97 and 109% at a 95% confidence interval, with precision of 4-9%. Concentrations of arsenic in the spiked drinking water, tap water, and pond water samples monitored by the developed method agreed well with conventional inductively coupled plasma optical emission spectrometry. This assay showed promise for on-site quantitative analysis of arsenic in water samples.

Mickey mouse-shaped laminated paper-based analytical device in simultaneous total cholesterol and glucose determination in whole blood.

The microfluidic paper-based analytical device (µPAD) platform is gaining attention as a low-cost, portable, and disposable detection tool. However, the limitations of traditional fabrication methods include poor reproducibility and the use of hydrophobic reagents. In this study, an in-house computer-controlled X-Y knife plotter and pen plotter were used to fabricate µPADs, resulting in a simple, more rapid, reproducible process that consumes less volume of reagents. The µPADs were laminated to increase mechanical strength and reduce sample evaporation during analysis. The resulting laminated paper-based analytical device (LPAD) was used to simultaneously determine glucose and total cholesterol in whole blood using the LF1 membrane as a sample zone. The LF1 membrane selectively separates plasma from whole blood by size exclusion and yields plasma for further enzymatic reaction steps while retaining blood cells and larger proteins. The i1 Pro 3 mini spectrophotometer directly detected color on the LPAD. The results were clinically relevant and in agreement with hospital methods, with a detection limit of 0.16 mmol L⁻1 for glucose and 0.57 mmol L⁻1 for TC. The LPAD retained color intensity after 60 days of storage. The LPAD offers a low-cost, high-performance option for chemical sensing devices and expands the applicability of markers for diagnosing whole blood samples.

A simple aptamer/gold nanoparticle aggregation-based colorimetric assay for oxidized low-density lipoprotein determination.

Oxidized low-density lipoprotein (oxLDL) is the leading cause of atherosclerosis and cardiovascular diseases. Here, we created a simple colorimetric assay for sensitive and specific determination of oxLDL using a selective aptamer coupled with salt-induced gold nanoparticle (AuNP) aggregation. The aptamer was chosen by Systematic Evolution of Ligands by Exponential Enrichment to obtain a novel selective sequence towards oxLDL (as 5'-CCATCACGGGGCAGGCGGACAAGGGGTAAGGGCCACATCA-3'). Mixing a 5 µM aptamer solution with an aliquot of a sample containing oxLDL followed by adding AuNP solution (OD = 1) and 80 mmol L-1 NaCl achieved rapid results within 19 min: linear response to oxLDL from 0.002 to 0.5 µmol L-1 with high selectivity, a recovery accuracy of 100-111% at the 95% confidence interval, and within-run and between-run precision of 1-6% and 1-5% coefficient variations, respectively. Artificial serum diluted at least 1:8 with distilled water, analyzed by the aptamer-based colorimetric assay, showed excellent correlation with conventional thiobarbituric acid reactive substances (TBARS) (R2 = 0.9792) as a rapid colorimetric method without the need for sample preparation other than dilution.

Enhanced oral bioavailability from food protein nanoparticles: A mini review.

The oral route is the most desirable drug administration path. The oral bioavailability is always compromised from the poor physicochemical and/or biopharmaceutical properties of the active pharmaceutical ingredients. Food protein nanoparticles show promise for oral drug delivery, with improved biosafety and cost-effectiveness compared to polymeric nanoparticles. More importantly, diverse food proteins provide "choice and variety" to meet the challenges faced by different drugs in oral delivery resulting from low solubility, poor permeability, and gastrointestinal stability. The abundance of hydroxyl, amino, and carboxyl groups in food proteins allows easy surface modification of the nanoparticles to impart unique functions. Albeit being in its infancy, food protein nanoparticles exhibit high capability to enhance oral bioavailability of a wide range of drugs from small molecules to biomacromolecules. Considering the rapid growth of the field, the achievements and mechanisms of food protein nanoparticles in enhancing oral bioavailability are reviewed. Factors affecting the performance of food protein nanoparticles are discussed with the purpose to inspire the development of food protein nanoparticle-based oral drug delivery systems.

Facile and Compact Electrochemical Paper-Based Analytical Device for Point-of-Care Diagnostic of Dual Carcinogen Oxidative Stress Biomarkers through a Molecularly Imprinted Polymer Coated on Graphene Quantum-Dot Capped Gold.

Nanoscale imprinting significantly increases the specific surface area and recognition capabilities of a molecularly imprinted polymer by improving accessibility to analytes, binding kinetics, and template removal. Herein, we present a novel synthetic route for a dual molecularly imprinted polymer (dual-MIP) of the carcinogen oxidative stress biomarkers 3-nitrotyrosine (3-NT) and 4-nitroquinolin-N-oxide (4-NQO) as coatings on graphene quantum-dot capped gold nanoparticles (GQDs-AuNPs). The dual-MIP was successfully coated on the GQDs-AuNPs core via a (3-mercaptopropyl) trimethoxysilane (MPTMS) linkage and copolymerization with the 3-aminopropyltriethoxysilane (APTMS) functional monomer. In addition, we fabricated a facile and compact three-dimensional electrochemical paper-based analytical device (3D-ePAD) for the simultaneous determination of the dual biomarkers using a GQDs-AuNPs@dual-MIP-modified graphene electrode (GQDs-AuNPs@dual-MIP/SPGE). The developed dual-MIP device provides greatly enhanced electrochemical signal amplification due to the improved electrode-specific surface area, electrocatalytic activity, and the inclusion of large numbers of dual-imprinted sites for 3-NT and 4-NQO detection. Quantitative analysis used square wave voltammetry, with an oxidation current appearing at -0.10 V for 4-NQO and +0.78 V for 3-NT. The dual-MIP sensor revealed excellent linear dynamic ranges of 0.01 to 500 µM for 3-NT and 0.005 to 250 µM for 4-NQO, with detection limits in nanomolar levels for both biomarkers. Furthermore, the dual-MIP sensor for the simultaneous determination of 3-NT and 4-NQO provides high accuracy and precision, with no evidence of interference from urine, serum, or whole blood samples.

Smart sensor for assessment of oxidative/nitrative stress biomarkers using a dual-imprinted electrochemical paper-based analytical device.

We present a novel dual-imprinted electrochemical paper-based analytical device (Di-ePAD) to simultaneously determine 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 3-nitrotyrosine (3-NT) and assess oxidative and nitrative biomarkers in urine and plasma samples. The Di-ePAD was designed with hydrophobic barrier layers formed on filter paper to provide three-dimensional circular reservoirs and assembled electrodes. The molecularly imprinted polymer (MIP) was synthesized using a silica nanosphere decorated with silver nanoparticles (SiO2@AgNPs) as a core covered with dual-analyte imprinted sites on the polymer to recognize selectively and bind the target biomarkers. This strategy drives monodispersity and enhances the conductivity of the resulting MIP core-shell products. 3-NT-MIP and 8-OHdG-MIP were synthesized by successively coating the surface of SiO2@AgNPs with l-Cysteine via the thiol group, then terminating with MIP shells. The dual imprinted core-shell composites possess attractive properties for the target biomarkers' sensing, including catalytic activity, selectivity, and good conductivity. The Di-ePAD revealed excellent linear dynamic ranges of 0.01-500 µM for 3-NT and 0.05-500 µM for 8-OHdG, with detection limits of 0.0027 µM for 3-NT and 0.0138 µM for 8-OHdG. This newly developed method based on the synergistic effects of SiO2@AgNPs combined with promising properties of MIP offers outstanding selectivity, sensitivity, reproducibility, simplicity, and low cost for quantitative analysis of 3-NT and 8-OHdG. The proposed Di-ePAD showed good accuracy and precision when applied to actual samples, including urine and serum samples validated by a conventional HPLC method.

Nitrogen-doped carbon dots/Ni-MnFe-layered double hydroxides (N-CDs/Ni-MnFe-LDHs) hybrid nanomaterials as immunoassay label for low-density lipoprotein detection.

Nitrogen-doped carbon dots/Ni-MnFe-layered double hydroxides (N-CDs/Ni-MnFe-LDHs) are demonstrated as superior peroxidase mimic antibody labels alternative to horseradish peroxidase (HRP) in an immunoassay, potentially overcoming some of the inherent disadvantages of HRP and other enzyme mimicking nanomaterials. They revealed efficient peroxidase-like activity and catalyzed the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to form the intense blue product (at 620 nm) in the presence of hydrogen peroxide (H2O2). Using low-density lipoprotein (LDL) as a model target, an ultra-low limit of detection (0.0051 mg/dL) and a linear range of 0.0625-0.750 mg/dL were achieved, exhibiting higher sensitivity than the HRP-based immunoassay. Thus, the proposed N-CDs/Ni-MnFe-LDHs can be used as HRP mimicking analogs for developing highly sensitive colorimetric immunosensors for detection of biomarkers, as well as trace chemical analysis.

Enhancing sensitivity of QCM for dengue type 1 virus detection using graphene-based polymer composites.

Graphene oxide-molecularly imprinted polymer composites (GO-MIP) have attracted significant attention as recognition materials in sensing due to their outstanding properties in terms of electrical and thermal conductivity, high mechanical modulus, and the comparably straightforward way to functionalize them. The aim of this study was to design a MIP-based sensor recognition material and enhance its sensitivity by blending it with GO for sensing a harmful dengue hemorrhagic fever pathogen, namely the dengue type 1 virus (DENV-1). Polymer composites comprising GO incorporated to an acrylamide (AAM)/methacrylic acid (MAA)/methyl methacrylate (MMA)/N-vinylpyrrolidone (VP) copolymer were synthesized and compared to the "pure" MIP, i.e., the copolymer without GO. The pure polymer revealed a zeta potential of + 9.9 ± 0.5 mV, whereas GO sheets prepared have a zeta potential of - 60.3 ± 2.7 mV. This results in an overall zeta potential of - 11.2 ± 0.2 mV of the composite. Such polymer composites seem appropriate to bind the positively charged DENV-1 particle (+ 42.2 ± 2.8 mV). GO-MIP coated onto 10-MHz quartz crystal microbalance (QCM) sensors indeed revealed two times sensitivity compared to sensors based on the pure MIP. Furthermore, GO-polymer composites revealed imprinting factors of up to 21, compared to 3 of the pure MIP. When plotting the sensor characteristic in a semilogarithmic way, the composite sensor shows the linear response to DENV-1 in the concentration range from 100 to 103 pfu mL-1. The corresponding limits of detection (S/N = 3) and quantification (S/N = 10) are 0.58 and 1.94 pfu mL-1, respectively. Furthermore, imprinted polymer composites selectively bind DENV-1 without significant interference: DENV-2, DENV-3, DENV-4, respectively, yield 13-16% of DENV-1 signal. The sensor requires only about 15-20 min to obtain a result.

Thin Film Plastic Antibody-Based Microplate Assay for Human Serum Albumin Determination.

Herein we demonstrate molecularly imprinted polymers (MIP) as plastic antibodies for a microplate-based assay. As the most abundant plasma protein, human serum albumin (HSA) was selected as the target analyte model. Thin film MIP was synthesized by the surface molecular imprinting approach using HSA as the template. The optimized polymer consisted of acrylic acid (AA) and N-vinylpyrrolidone (VP) in a 2:3 (w/w) ratio, crosslinked with N,N'-(1,2-dihydroxyethylene) bisacrylamide (DHEBA) and then coated on the microplate well. The binding of MIP toward the bound HSA was achieved via the Bradford reaction. The assay revealed a dynamic detection range toward HSA standards in the clinically relevant 1-10 g/dL range, with a 0.01 g/dL detection limit. HSA-MIP showed minimal interference from other serum protein components: γ-globulin had 11% of the HSA response, α-globulin of high-density lipoprotein had 9%, and ß-globulin of low-density lipoprotein had 7%. The analytical accuracy of the assay was 89-106% at the 95% confidence interval, with precision at 4-9%. The MIP-coated microplate was stored for 2 months at room temperature without losing its binding ability. The results suggest that the thin film plastic antibody system can be successfully applied to analytical/pseudoimmunological HSA determinations in clinical applications.

Mimicking Peroxidase-Like Activity of Nitrogen-Doped Carbon Dots (N-CDs) Coupled with a Laminated Three-Dimensional Microfluidic Paper-Based Analytical Device (Laminated 3D-µPAD) for Smart Sensing of Total Cholesterol from Whole Blood.

This work presents a simple hydrothermal synthesis of nitrogen-doped carbon dots (N-CDs), fabrication of microfluidic paper-based analytical device (µPAD), and their joint application for colorimetric determination of total cholesterol (TC) in human blood. The N-CDs were characterized by various techniques including transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and X-ray powder diffraction (XRD), and the optical and electronic properties of computational models were studied using the time-dependent density functional theory (TD-DFT). The characterization results confirmed the successful doping of nitrogen on the surface of carbon dots. The N-CDs exhibited high affinity toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS) with the Michaelis-Menten constant (KM) of 0.018 mM in a test for their peroxidase-like activity. Particularly, since hydrogen peroxide (H2O2) is the oxidative product of cholesterol in the presence of cholesterol oxidase, a sensitive and selective method of cholesterol detection was developed. Overall, the obtained results from TD-DFT confirm the strong adsorption of H2O2 on the graphitic N positions of the N-CDs. The laminated three-dimensional (3D)-µPAD featuring a 6 mm circular detection zone was fabricated using a simple wax screen printing technique. Classification of TC according to the clinically relevant criteria (healthy, <5.2 mM; borderline, 5.2-6.2 mM; and high risk, >6.2 mM) could be determined by the naked eye within 10 min by simple comparison using a color chart. Overall, the proposed colorimetric device serves as a low-cost, rapid, simple, sensitive, and selective alternative for TC detection in whole blood samples that is friendly to unskilled end users.

Direct assessment of very-low-density lipoprotein by mass sensitive sensor with molecularly imprinted polymers.

Very-low-density lipoprotein (VLDL) contributes to the buildup of atherosclerotic plaque in the arteries and can lead to coronary heart disease. In clinical laboratory testing, the cholesterol content of VLDL (VLDL-C) cannot be assessed directly by the enzymatic colorimetric assay as it can for other lipoproteins, due to lack of a specific sample pretreatment technique. VLDL concentration relies on analyzing the endogenous triglycerides (TGs) bound in its particles and then converting to the VLDL-C estimate TGs/5. This estimation is valid for at least 12 h-fasted serum when exogenous TGs attached to chylomicrons (CMs) have been cleared from the circulation. A quartz crystal microbalance (QCM)-based sensor was generated using biomimetic sensing elements as a molecularly imprinted polymer (MIP) to directly measure actual VLDL. A novel VLDL-MIP was synthesized using methacrylic acid (MAA) and N-vinylpyrrolidone (VP) in the ratio 1:1 (v/v) as functional monomers in the presence of N, N'-(1,2-dihydroxyethylene) bis(acrylamide) (DHEBA) as a crosslinking agent. The VLDL-MIP sensor showed high sensitivity with a linear response from 2.5 mg dL-1 to 100 mg dL-1 of VLDL-C with a limit of detection at 1.5 mg dL-1. Recoveries of 96-103% were achieved when the VLDL-MIP sensor was used for VLDL assessment at 38-71 mg dL-1 concentrations. Repeatability and reproducibility of the sensor were very good with coefficients of variation at 1.63-4.74% and 4.25-9.04%, respectively. The sensor demonstrated low cross-reactivity with other lipoproteins; 6-7% of low-density lipoprotein (LDL) signals, 2-4% high-density lipoprotein (HDL), and 1% CMs compared to the signal of VLDL. Sensor results for 12 h-fasted serum and non-fasted serum correlated well with VLDL estimates TGs/5, with coefficients of determination (R2) at 0.9967 and 0.9932, respectively. This new sensor offers a new strategy for direct VLDL assessment from non-fasted serum without other sample pretreatment steps than dilution.

Biomimetic sensors targeting oxidized-low-density lipoprotein with molecularly imprinted polymers.

Oxidized-low-density lipoprotein (oxLDL) is well-recognized as an actual patho-atherogenic lipoprotein: elevated serum concentration of oxLDL increases the risk for developing atherosclerosis, leading to coronary artery disease (CAD). Herein, we report an approach for sensing oxLDL directly in serum with molecularly imprinted polymer (MIP) thin films on quartz crystal microbalance (QCM). The resulting MIP sensors show low cross-reaction toward low-density lipoprotein (LDL) and high-density lipoprotein (HDL): signals are around one magnitude smaller. Very-low-density lipoprotein (VLDL) and human serum albumin (HSA) do not lead to any significant sensor response. The sensor allowed for accurately assessing oxLDL over the detection range of 86-5600 µg dL-1, which covers the clinically relevant concentrations. The sensor determines oxLDL with recovery accuracy of 92-107% and a precision of 1-8% coefficient variation. Compared with commercially available oxLDL ELISA test kit our sensor reveals similar characteristics obtaining a correlation coefficient of 0.98. However, the sensors have rapid response times of 10 min compared to 210 min of ELISA, which demonstrates their efficiency in assessing this sensitive atherogenic biomarker for CAD diagnostics.

High-density lipoprotein sensor based on molecularly imprinted polymer.

Decreased blood level of high-density lipoprotein (HDL) is one of the essential criteria in diagnosing metabolic syndrome associated with the development of atherosclerosis and coronary heart disease. Herein, we report the synthesis of a molecularly imprinted polymer (MIP) that selectively binds HDL, namely, HDL-MIP, and thus serves as an artificial, biomimetic sensor layer. The optimized polymer contains methacrylic acid and N-vinylpyrrolidone in the ratio of 2:3, cross-linked with ethylene glycol dimethacrylate. On 10 MHz dual electrode quartz crystal microbalances (QCM), such HDL-MIP revealed dynamic detection range toward HDL standards in the clinically relevant ranges of 2-250 mg/dL HDL cholesterol (HDL-C) in 10 mM phosphate-buffered saline (PBS, pH = 7.4) without significant interference: low-density lipoprotein (LDL) yields 5% of the HDL signal, and both very-low-density lipoprotein (VLDL) and human serum albumin (HSA) yield 0%. The sensor reveals recovery rates between 94 and 104% at 95% confidence interval with precision of 2.3-7.7% and shows appreciable correlation (R 2 = 0.97) with enzymatic colorimetric assay, the standard in clinical tests. In contrast to the latter, it achieves rapid results (10 min) during one-step analysis without the need for sample preparation. Graphical Abstract ᅟ.

Low-Density Lipoprotein Sensor Based on Molecularly Imprinted Polymer.

Increased level of low-density lipoprotein (LDL) strongly correlates with incidence of coronary heart disease. We synthesized novel molecularly imprinted polymers (MIP) as biomimetic specific receptors to establish rapid analysis of LDL levels. For that purpose the ratios of monomers acrylic acid (AA), methacrylic acid (MAA), and N-vinylpyrrolidone (VP), respectively, were screened on 10 MHz dual-electrode quartz crystal microbalances (QCM). Mixing MAA and VP in the ratio 3:2 (m/m) revealed linear sensor characteristic to LDL cholesterol (LDL-C) from 4 to 400 mg/dL or 0.10-10.34 mmol/L in 100 mM phosphate-buffered saline (PBS) without significant interference: high-density lipoprotein (HDL) yields 4-6% of the LDL signal, very-low-density-lipoprotein (VLDL) yields 1-3%, and human serum albumin (HSA) yields 0-2%. The LDL-MIP sensor reveals analytical accuracy of 95-96% at the 95% confidence interval with precision at 6-15%, respectively. Human serum diluted 1:2 with PBS buffer was analyzed by LDL-MIP sensors to demonstrate applicability to real-life samples. The sensor responses are excellently correlated to the results of the standard technique, namely, a homogeneous enzymatic assay (R(2) = 0.97). This demonstrates that the system can be successfully applied to human serum samples for determining LDL concentrations.

A piezoelectric-based immunosensor for high density lipoprotein particle measurement.

A piezoelectric-based immunosensor was developed for high density lipoprotein particle (HDL-P) measurement. Monoclonal anti-human apolipoprotein A1 antibody was used as a specific binding molecule for the major apolipoprotein of HDL-P. This sensing element was fabricated by immobilizing the anti-human apolipoprotein A1 on a 12 MHz AT-cut quartz crystal via a 3-mercaptopropionic acid (MPA) self-assembled monolayer. The frequency shift from the mass change of the antigen-antibody binding refers to the amount of HDL-P. The optimal antibody immobilization was performed to achieve the maximum potential of the antibody. The appropriate quantity and immobilization time of the antibody were 0.1 mg ml(-1) and 90 minutes, respectively. The immobilized antibody in the HDL-P immunosensor accomplished perfect binding with HDL-P within 60 minutes. The dose-response curve for HDL-P showed a linear response from 0.21 to 2.50 mg protein per ml equivalent to 0.40 × 10(10) to 3.65 × 10(10) particles per µl without significant interference from other lipoproteins. The intra- and inter-assay imprecision (CV) were 7.8 and 18.5%, respectively. The analytical accuracy of this measurement was 96.29-96.31%. The HDL-P concentration obtained from the sensor revealed a 2.05 mg protein per ml with 0.26 mg protein per ml of expanded uncertainty at the 95% confidence level. This immunosensor gave an assay result which correlated with the homogeneous enzymatic colorimetric assay (R(2) = 0.902).