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Summary: Total antioxidant capacity (TAC), representative of the capacity to combat oxidative stress, is closely linked to numerous diseases. Here, we present a protocol for measuring TAC using minimal samples that are stable across varying pH levels and at room temperature. We describe steps for preparing and loading samples and working solutions and conducting and analyzing the colorimetric reaction. Sample sources include aqueous humor, vitreous, tears, and plasma, which allow the protocol to be used in various clinical diagnostic settings.For complete details on the use and execution of this protocol, please refer to publications by Tsao et al. (2022).1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Biochemical analysis of human body fluids is a frequent and fruitful strategy for disease diagnosis. Point-of-care (POC) diagnostics offers the tantalizing possibility of providing rapid diagnostic results in non-laboratory settings. Successful diagnostic testing using body fluids has been reported on in the literature; however, small-volume detection devices, which offer remarkable advantages such as portability, inexpensiveness, capacity for mass production, and tiny sample volume requirements have not been thoroughly discussed. Here, we review progress in this research field, with a focus on developments since 2015. In this review article, we provide a summary of articles that have detailed the development of small-volume detection strategies using clinical samples over the course of the last 5 years. Topics covered include small-volume detection strategies in ophthalmology, dermatology or plastic surgery, otolaryngology, and cerebrospinal fluid analysis. In ophthalmology, advances in technology could be applied to examine tear or anterior chamber (AC) fluid for glucose, lactoferrin, interferon, or VEGF. These approaches could impact detection and care for diseases including diabetic mellitus, dry-eye disease, and age-related maculopathy. Early detection and easy monitoring are critical approaches for improving overall care and outcome. In dermatology or plastic surgery, small-volume detection strategies have been applied for passive or interactive wound dressing, wound healing monitoring, and blister fluid analysis for autoimmune disease diagnosis. In otolaryngology, the analysis of nasal secretions and mucosa could be used to differentiate between allergic responses and infectious diseases. Cerebrospinal fluid analysis could be applied in neurodegenerative diseases, central neural system infection and tumor diagnosis. Other small-volume fluids that have been analyzed for diagnostic and monitoring purposes include semen and cervico-vaginal fluids. We include more details regarding each of these fluids, associated collection and detection devices, and approaches in our review.

Abstract Corneal endothelial decompensation is a serious condition that frequently requires treatment via corneal transplantation which contributes to a global shortage in donor corneas. Therefore, the purpose of this study was to analyze the influence of aqueous humor total antioxidant capacity (TAC) on corneal endothelial health. There is an urgent need for discovering protective factors to combat corneal endothelial cell (CEC) loss. For methods, we developed a cupric ion‐based TAC (CuTAC) assay to analyze TAC level in a small volume of aqueous humor, that is, 10 μL per test, and examined the influences of ascorbic acid (AA) and antioxidant proteins on aqueous humor TAC. To broaden the investigation, we conducted a case–control study with patients classified into two groups, an insufficient endothelial cell density (ECD

Abstract Male infertility affects millions of males worldwide and is rising in prevalence due to social and environmental conditions. However, men often feel too embarrassed to receive a semen analysis in the hospital due to social stigmas. To overcome this problem, we developed a 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide test strip to distinguish semen samples with low total motile sperm concentration from those with normal motile sperm concentration. This is a point‐of‐care colorimetric semen analytical method with a one‐step, inexpensive, equipment‐free evaluation process, and adequate accuracy validated in a 42‐sample clinical trial. In this study, results were evaluated visually and with a smartphone application. Using visual observation methods, the area under the curve (AUC) was 0.71 (95% of confidence interval = 0.55–0.86; p = 0.021), sensitivity was 41%, specificity was 95%, positive predictive value was 90%, negative predictive value (NPV) was 59.4%, and accuracy was 67%. Using a smartphone recording and analytical system, AUC was 0.766 (95% of confidence interval = 0.612–0.92; p = 0.003), sensitivity was 96%, specificity was 65%, PPV was 75%, NPV was 92.9%, and accuracy was 80.9%. This work demonstrated a screening tool that could elevate semen analysis to the level of routine healthcare and provide for private, in‐home self‐assessment.

Cataract is the leading cause of blindness throughout the world. Currently, the cataract severity evaluation is based on the subjective LOCS III guideline. To ameliorate the evaluation system and develop an objective and quantitative analysis, we investigated the relationships among aqueous humor total antioxidant capacity (AqTAC), ascorbic acid (AqAA) concentration, and cataract severity. In this study, we enrolled 130 cataract patients who underwent phacoemulsification between April 2019 and March 2020. The AqTAC and AqAA were measured by our own developed TAC assay and commercially available kit. Cataract severity was recorded by nuclear opalescence (NO) and cortical cataract (CC) degree according to LOCS III. Cumulative dissipated energy (CDE) during phacoemulsification was recorded to verify the severity of the cataract. As a result, we found a moderate correlation between AqTAC and CDE (p < 0.001). In addition, we found AqTAC independently associated with the CDE when analyzed by multivariate linear regression (p < 0.001). AqTAC also negatively correlated to cataract severity when measured by NO and CC (p = 0.012 in NO grade 3 vs. grade 1; p = 0.012 in CC grade 2 vs. grade 1; p < 0.001 in CC grade 3 vs. grade 1). We further found AqAA provided 71.9 ± 13.5% of AqTAC, and showed a high correlation (rho = 0.79, p < 0.001). In conclusion, we found a significant correlation between AqTAC/AqAA and cataract severity measured by CDE. The correlation was superior to the correlation between LOCS III and CDE. Aqueous humor TAC owns the potential to assess cataracts in an objective and quantitative way.

Oxidative stress is an important pathomechanism found in numerous ocular degenerative diseases. To provide a better understanding of the mechanism and treatment of oxidant/antioxidant imbalance-induced ocular diseases, this article summarizes and provides updates on the relevant research. We review the oxidative damage (e.g., lipid peroxidation, DNA lesions, autophagy, and apoptosis) that occurs in different areas of the eye (e.g., cornea, anterior chamber, lens, retina, and optic nerve). We then introduce the antioxidant mechanisms present in the eye, as well as the ocular diseases that occur as a result of antioxidant imbalances (e.g., keratoconus, cataracts, age-related macular degeneration, and glaucoma), the relevant antioxidant biomarkers, and the potential of predictive diagnostics. Finally, we discuss natural antioxidant therapies for oxidative stress-related ocular diseases.

Early detection of microorganisms is essential for the management of infectious diseases. However, this is challenging, as traditional culture methods are labor-intensive and time-consuming. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-phenazine methosulfate (MTT-PMS) assay has been used to evaluate the metabolic activity in live cells and can thus be used for detecting living microorganisms. With the addition of NaOH and Tris-EDTA, the same approach can be accelerated (within 15 min) and used for the quick detection of common bacterial pathogens. The assay results can be evaluated colorimetrically or semi-quantitatively. Here, the quick detection by MTT-PMS assay was further investigated. The assay had a detection limit of approximately 104 CFU/mL. In clinical evaluations, we used the MTT-PMS assay to detect clinical samples and bacteriuria (>105 CFU/mL). The negative predictive value of the MTT-PMS assay for determining bacteriuria was 79.59% but was 100% when the interference of abnormal blood was excluded. Thus, the MTT-PMS assay might be a potential “rule-out” tool for bacterial detection in clinical samples, at a cost of approximately USD 1 per test. Owing to its low cost, rapid results, and easy-to-use characteristics, the MTT-PMS assay may be a potential tool for microorganism detection.

Urinary tract infections (UTI), one of the most common bacterial infections, annually affect 150 million people worldwide. Infants and the elderly are likely to have missed or delayed diagnosis of UTI due to difficulty clearly describing their symptoms. A rapid screening method for UTI is a critical and urgent need for these populations. The aim of our study is to develop a diaper-based testing device to assay urine biomarkers including pH, leukocyte, and nitrite level. This all-in-one device assists in urine collection and testing using a colorimetric approach to provide easily read visual results on the outside surface of a test strip-integrated diaper. In this study, we tested samples from 46 patients using testing strips and examined the results from 7 patients recruited to validate the strip-integrated diaper. In conclusion, this new diaper-based testing device is easy to use, rapid, and inexpensive, all of which imbue it with tremendous potential for development into a commercially viable UTI screening system.