Simultaneous Detection of Serum Glucose and Glycated Albumin on a Paper-Based Sensor for Acute Hyperglycemia and Diabetes Mellitus.

Diabetes mellitus is one of the most common chronic diseases worldwide. Generally, the levels of fasting or postprandial blood glucose and other biomarkers, such as glycated albumin, glycated hemoglobin, and 1,5-anhydroglucitol, are used to diagnose or monitor diabetes progression. In the present study, we developed a sensor to simultaneously detect the glucose levels and glycation ratios of human serum albumin using a lateral flow assay. Based on the specific enzymatic reactions and immunoassays, a spiked glucose solution, total human serum albumin, and glycated albumin were measured simultaneously. To test the performance of the developed sensor, clinical serum samples from healthy subjects and patients with diabetes were analyzed. The glucose level and glycation ratios of the clinical samples were determined with reasonable correlation. The R-squared values of glucose level and glycation ratio measurements were 0.932 and 0.930, respectively. The average detection recoveries of the sensor were 85.80% for glucose and 98.32% for the glycation ratio. The glucose level and glycation ratio in our results were crosschecked with reference diagnostic values of diabetes. Based on the outcomes of the present study, we propose that this novel platform can be utilized for the simultaneous detection of glucose and glycation ratios to diagnose and monitor diabetes mellitus.

Paper-based 1,5-anhydroglucitol quantification using enzyme-based glucose elimination.

The monosaccharide 1,5-anhydroglucitol (1,5-AG) is a known indicator of glucose levels. Conventional 1,5-AG quantification methods with enzyme-based sensors using pyranose oxidase (PROD) require elimination of interference from the sample (a laborious and time-consuming process), as PROD cannot distinguish 1,5-AG from other sugars. We developed a one-step paper-based sensor for detecting 1,5-AG using glucose oxidase, catalase, and mutarotase that eliminates excess glucose, which interferes with 1,5-AG detection. This sensor consists of two compartments for the quantification of glucose and 1,5-AG and reflects the concentration of these targets after reaction with water or spiked human urine. The limit of detection of the sensor was 0.9 mg dL-1 for glucose and 3.2 µg mL-1 for 1,5-AG.

Glycation ratio determination through simultaneous detection of human serum albumin and glycated albumin on an advanced lateral flow immunoassay sensor.

Glycated albumin synthesized in a non-enzymatic reaction with high glucose levels in human plasma is a long-term biomarker for understanding average glucose levels over the past few weeks. Glycated albumin level determination requires an enzymatic assay involving an expensive, complicated, and laborious process, including the specific hydrolysis of albumin and the oxidation of glycated amino acids. In this study, we developed two advanced lateral flow immunoassays (LFIAs) for the simultaneous determination of total human serum albumin and glycated albumin concentrations using a colorimetric signal. Additionally, through a sequential reaction on our advanced LFIA, the selectivity of glycated albumin was improved. We quantified both HSA and GA with wide detection ranges of 1 ng mL-1-1 mg mL-1 and 0.5 µg mL-1-3.6 mg mL-1, respectively. Various serum samples with different glycation ratios were analyzed using this sensor and demonstrated a reasonable recovery range. This indicated that our platform could directly determinate the glycation ratio of various samples, and therefore, be applicable in point-of-care glucose status monitoring.

Determination of cerebrospinal fluid leakage by selective deletion of transferrin glycoform using an immunochromatographic assay.

Cerebrospinal fluid (CSF) leakage can lead to brain and spine pathologies and there is an urgent need for a rapid diagnostic method for determining CSF leakage. Beta-2 transferrin (ß2TF), asialotransferrin, is a specific CSF glycoprotein biomarker used to determine CSF leakage when distinguished from serum sialotransferrin (sTF). Methods: We detected ß2TF using an immunochromatographic assay (ICA), which can be potentially developed as a point-of-care (POC) testing platform. Sialic acid-specific lectin selectively captures sTF in multiple deletion lines within an ICA test strip, enabling the detection of ß2TF. A sample pre-treatment process efficiently captures excess sTF increasing sensitivity for CSF leakage detection. Results: An optimal cut-off value for determining the presence of CSF in test samples was obtained from receiver operating characteristic (ROC) analysis of the ratio of the test signal intensity and the deletion lines. On 47 clinical samples, ICA test strips discriminated CSF positive from negative samples with statistically significant (positive versus negative t-test; P =0.00027). Additional artificial positive samples, prepared by mixing CSF positive and negative clinical samples, were used as a further challenge. These positive samples were clearly discriminated from the negative samples (mixture versus negative t-test; P =0.00103) and CSF leakage was determined with 97.1% specificity and 96.2% sensitivity. Conclusions: ICA represents a promising approach for POC diagnosis of CSF leakage. While requiring 70 min assay time inconvenient for POC testing, our method was significantly shorter than conventional electrophoresis-based detection methods for ß2TF.

Development of Replication Protein A-Conjugated Gold Nanoparticles for Highly Sensitive Detection of Disease Biomarkers.

Paper-based lateral flow immunoassays (LFIAs) using conventional sandwich-type immunoassays are one of the most commonly used point-of-care (PoC) tests. However, the application of gold nanoparticles (AuNPs) in LFIAs does not meet sensitivity requirements for the detection of infectious diseases or biomarkers present at low concentrations in body fluids because of the limited number of AuNPs that can bind to the target. To overcome this problem, we first developed a single-stranded DNA binding protein (RPA70A, DNA binding domain A of human Replication Protein A 70 kDa) conjugated to AuNPs for a sandwich assay using a capture antibody immobilized in the LFIA and an aptamer as a detection probe, thus, enabling signal intensity enhancement by attaching several AuNPs per aptamer. We applied this method to detect the influenza nucleoprotein (NP) and cardiac troponin I (cTnI). We visually detected spiked targets at a low femtomolar range, with limits of detection for NP in human nasal fluid and for cTnI in serum of 0.26 and 0.23 pg·mL-1, respectively. This technique showed significantly higher sensitivity than conventional methods that are widely used in LFIAs involving antibody-conjugated AuNPs. These results suggest that the proposed method can be universally applied to the detection of substances requiring high sensitivity and can be used in the field of PoC testing for early disease diagnosis.

Advanced Colorimetric Paper Sensors Using Color Focusing Effect Based on Asymmetric Flow of Fluid.

Although paper-based colorimetric sensors utilizing enzymatic reactions are well suited for real-field diagnosis, their widespread use is hindered by signal blurring at the detection spot due to the action of capillary forces on the liquid and the corresponding membrane. In this study, we eliminated signal losses commonly observed during enzyme-mediated colorimetric sensing and achieved pattern-free quantitative analysis of glucose and uric acid by mixing enzymes and color-forming reagents with chitosan oligosaccharide lactate (COL), which resulted in perfectly focused colorimetric signals at the detection spot, using asymmetric flow induced by changing the flow rate of the COL-treated paper. The targets were calibrated with 0-500 mg/dL of glucose and 0-200 mg/dL of uric acid, and the limit of detection was calculated to be 0.6 and 0.03 mg/dL, respectively. In human urine, the correlation has a high response between the measured and spiked concentrations, and the stability of the enzyme mixture including COL increased by 41% for glucose oxidase mixture and 29% for uricase mixture, compared to the corresponding mixtures without COL. Thus, the color focusing and pattern-free sensor, which have the advantages of easy fabrication, easy handling, and high stability, should be applied to real-field diagnosis.

A hook effect-free immunochromatographic assay (HEF-ICA) for measuring the C-reactive protein concentration in one drop of human serum.

The immunochromatographic (ICA) assay is a highly promising platform for rapid and simple detection of C-reactive protein (CRP) which is an indicator of the different phases of various diseases, as well as of inflammation and infection. However, the hook effect in the ICA assay limits the quantification of CRP levels at high CRP concentrations. Methods: In this study, we developed a hook effect-free immunochromatographic assay (HEF-ICA) to detect CRP over a wide concentration range. The hook effect results from the simultaneous reaction of an excess target antigens with both immobilized and labeled antibodies respectively. To reduce the potential occurrence of this simultaneous reaction, we separated the migration of the target antigen and gold nanoparticle (AuNP)-labeled antibodies on a nitrocellulose membrane and analyzed the time profiles by modifying the ICA structure. Results: The signal intensity of HEF-ICA was saturated at high CRP concentrations, without decreasing. The titration curve of HEF-ICA was adjusted with the Hill equation, and HEF-ICA was performed with the following parameters: limit of detection, 43 ng mL-1; dynamic range, 119 ng mL-1 to 100 µg mL-1. The accuracy of the newly developed assay was evaluated using 33 clinical samples via comparison with a clinical chemistry analyzer. Conclusion: HEF-ICA enabled the measurement of a wide range of CRP concentrations without the hook effect, and was suitable for point-of-care testing with fingertip blood sampling, as only a minute sample volume (2.5 µL) was required.