Point-of-care blood tests using a smartphone-based colorimetric analyzer for health check-up.

A microscale colorimetric assay was designed and implemented for the simultaneous determination of clinical chemistry tests measuring six parameters, including glucose (GLU), total protein (TP), human serum albumin (HSA), uric acid (UA), total cholesterol (TC), and triglycerides (TGs) in plasma samples. The test kit was fabricated using chromogenic reagents, comprising specific enzymes and binding dyes. Multiple colors that appeared on the reaction well when it was exposed to each analyte were captured by a smartphone and processed by the homemade Check6 application, which was designed as a colorimetric analyzer and simultaneously generated a report that assessed test results against gender-dependent reference ranges. Six blood checkup parameters for four plasma samples were conducted within 12 min on one capture picture. The assay achieved wide working concentration ranges of 10.45-600 mg dL-1 GLU, 1.39-10.0 g dL-1 TP, 1.85-8.0 g dL-1 HSA, 0.86-40.0 mg dL-1 UA, 11.28-600 mg dL-1 TC, and 11.93-400 mg dL-1 TGs. The smartphone-based assay was accurate with recoveries of 93-108% GLU, 93-107% TP, 92-107% HSA, 93-107% UA, 92-107% TC, and 99-113% TGs. The coefficient of variation for intra-assay and inter-assay precision ranged from 3.2-5.2% GLU, 4.6-5.3% TP, 4.3-5.3% HSA, 2.8-6.6% UA, 2.7-6.5% TC, and 1.1-3.9% TGs. This assay demonstrated remarkable accuracy in quantifying the concentration-dependent color intensity of the plasma, even in the presence of other suspected interferences commonly present in serum. The results of the proposed method correlated well with results determined by the microplate spectrophotometer (R2 > 0.95). Measurement of these six clinical chemistry parameters in plasma using a microscale colorimetric test kit coupled with the Check6 smartphone application showed potential for real-time point-of-care analysis, providing cost-effective and rapid assays for health checkup testing.

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.

Vanadium-Doped Porous Cobalt Oxide for Its Superior Peroxidase-like Activity in Simultaneous Total Cholesterol and Glucose Determination in Whole Blood Based on a Simple Two-Dimensional Paper-Based Analytical Device.

Vanadium-doped porous Co3O4 (V-porous Co3O4) was synthesized via a simple soft-templating method and used as a superior peroxidase mimic for the simultaneous colorimetric determination of glucose and total cholesterol (TC) in whole blood samples on a two-dimensional microfluidic paper-based analytical device (2D-µPAD). The large surface area and the presence of two metals in V-porous Co3O4 contributed to its excellent catalytic activity toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 3,3',5,5'- tetramethylbenzidine (TMB) with Michaelis-Menten constants (KM) of 0.1301 and 0.0141 mM, respectively. The 2D-µPAD was fabricated using simple wax screen-printing and cutting techniques. The colorimetric reactions of both glucose and TC on 2D-µPAD were simultaneously performed by adding a single drop of a whole blood sample on the sample zone made of the LF1 membrane. After the enzymatic reactions, the generated hydrogen peroxide (H2O2) was oxidized by V-porous Co3O4 to produce hydroxy radicals (•OH), inducing ABTS and TMB to generate colored products. The generated H2O2 was proportional to the intensities of the green and blue products of the glucose and TC systems, respectively. The developed 2D-µPAD required a short analysis time (∼5 min) with small volumes of samples (15 µL of whole blood) whereby no sample preparation was needed. Owing to several advantages including simplicity, low cost, long-term stability, and simultaneous readout, the novel V-porous Co3O4 coupled with 2D-µPAD proved to be promising for practical uses as a pioneering portable device for the determination of glucose, TC, and other important biomarkers without the need of technical supports.

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.

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.

Acid-base titration using a microfluidic thread-based analytical device (µTAD).

This work presents the development and application of a novel analytical approach for the determination of acid and base concentrations by titration using a microfluidic thread-based analytical device (µTAD). This approach proved to be a simple to fabricate and to use, high precision, and cost-efficient means of acid-base quantification. The µTAD was fabricated by immobilizing the untreated cotton threads onto a wood frame, followed by pre-coating with an indicator (20 µL) and a primary standard solution (3 µL), and was tested using real samples including drug, food, and household products where 3 µL of each sample was dropped onto the center of a thread. Afterward, the distance of color change on the thread, easily observed and measured using the naked eye and a ruler, was used for analysis. The analysis using the µTAD, completed within 2 minutes and validated by the conventional titration, showed high accuracy and precision (RSD < 12.9%), good linearity ranges and low limit of quantification. The fabricated µTAD also remained stable for an extended period of time (>2 weeks under various storage conditions).