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.

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.

Deoxyribonucleic acid (DNA) extraction procedure is commonly based on well-developed and commercialized silica-based techniques. However, there are limits to utilize in resource-limited environments due to being time-consuming, labor-intensive, and expensive. Although simple DNA extraction systems have been recently reported, there are still remaining challenges for conventional treatment to complex samples such as blood, food, and soil. Here, we demonstrated a handheld lateral flow strip for rapid DNA extraction from complex samples. We designed the lateral flow system based on investigation of basic property for DNA binding and proper arrangements of various materials. Engineering structure of the lateral flow strip made by the arrangement of convenient pad materials is playing a key role for DNA extraction device without expensive fabrication technique. Different binding affinity to DNA molecules of pad materials is inducing DNA transportation and concentration on the elution hole by lateral flow separation. Result of rapid DNA extraction by the handheld strip was shown similar efficiency to a commercialized silica column. In the concentration between 10 CFU to 10 6 CFU, and the volume of 5 μl to 100 μl, DNA extraction from Staphylococcus aureus cells in buffer was successfully performed by the handheld strip. We successfully showed that the rapid and effective DNA extraction in various type of complex samples (such as saliva, whole blood, urine, milk, meat, soil, and pond water) can be performed using the handheld strip within 3 min. Moreover, this fabricated strip could be an excellent platform for DNA storage and transportation, thus being potentially suitable for expanding the area of DNA analysis in resourcelimited environments.

The detection of trace protein biomarkers is essential in the diagnostic field. Protein detection systems ranging from widely used enzyme-linked immunosorbent assays to simple, inexpensive approaches, such as lateral flow immunoassays, play critical roles in medical and drug research. Despite continuous progress, current systems are insufficient for the diagnosis of diseases that require high sensitivity. In this study, we developed a heterogeneous sandwich-type sensing platform based on recombinase polymerase amplification using DNA aptamers specific to the target biomarker. Only the DNA bound to the target in the form of a heterogeneous sandwich was selectively amplified, and the fluorescence signal of an intercalating dye added before the amplification reaction was detected, thereby enabling high specificity and sensitivity. We applied this method for the detection of protein biomarkers for various infectious diseases including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and observed attomolar-level detection of biomarkers and low cross-reactivity between different viruses. We also confirmed detection efficiency of the proposed method using clinical samples. These results demonstrate that the proposed sensing platform can be used to diagnose various diseases requiring high sensitivity, specificity, and accuracy.

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 colorforming 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.