RESEARCH AND DEVELOPMENT OF A SPECIFIC APTAMER-BASED KIT FOR DETECTING MICROSCOPIC HEMATURIA IN URINE

Description: Microhematuria is an important early indicator of urinary tract diseases, including urinary tract infections, kidney disorders, and bladder cancer. However, existing diagnostic methods have significant limitations. Microscopic urinalysis requires specialized laboratory equipment and trained personnel, whereas commercial urine dipstick tests are susceptible to interference from urine pH, vitamin C, and other oxidizing substances, reducing their diagnostic accuracy. This study aimed to develop a rapid, sensitive, and highly selective colorimetric biosensor based on a hemin-specific DNA aptamer for the detection of microhematuria. The proposed sensing platform utilizes the specific interaction between a DNA aptamer and hemin, the iron-containing component of hemoglobin. Upon binding, the aptamer stabilizes hemin and enhances its peroxidase-like catalytic activity, enabling efficient oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by hydrogen peroxide. The resulting color change provides a simple visual readout and can also be quantified spectrophotometrically at 450 nm. To maximize analytical performance, reaction conditions including pH, NaCl concentration, MgCl₂ concentration, TMB concentration, hydrogen peroxide concentration, incubation time, and aptamer structure were systematically optimized. Under the optimized conditions, the biosensor achieved a limit of detection (LOD) of 0.14 nM and a limit of quantification (LOQ) of 0.46 nM, which are substantially lower than the hemin concentrations typically associated with clinical microhematuria. The sensor also demonstrated stable performance in artificial urine and successfully detected trace amounts of sheep blood at concentrations representative of microscopic hematuria, producing results comparable to commercial urine dipsticks. This study demonstrates that aptamer-based molecular recognition can significantly improve the sensitivity and selectivity of colorimetric biosensors for urinary diagnostics. The developed platform provides a promising foundation for a low-cost, rapid, and portable diagnostic kit manufactured in Vietnam, with future potential for smartphone-assisted analysis and telehealth applications.

1. Background Microhematuria, defined as the presence of red blood cells in urine that cannot be observed with the naked eye, is an important clinical indicator of urinary tract disorders. It may result from relatively benign conditions such as urinary tract infections or kidney stones, but it can also represent an early warning sign of severe diseases including glomerulonephritis, bladder cancer, and kidney cancer. Because early-stage urinary diseases often present with few or no symptoms, reliable screening methods for microhematuria are essential for early diagnosis and timely treatment. Current diagnostic approaches mainly include microscopic urinalysis and commercial urine dipsticks. Although microscopic examination is considered the gold standard, it requires trained personnel, laboratory equipment, and time-consuming sample preparation. Dipstick tests are rapid and inexpensive but often suffer from false-positive and false-negative results because their chemical reactions are influenced by urine pH, vitamin C, oxidizing agents, and other interfering substances. Therefore, there is a need for a rapid, sensitive, selective, and affordable diagnostic platform. 2. Research Objective This study developed a colorimetric biosensor based on a hemin-specific DNA aptamer for detecting microhematuria in urine. Hemin, the iron-containing prosthetic group of hemoglobin, was selected as the detection target because every hemoglobin molecule contains four hemin groups and hemin possesses intrinsic peroxidase-like catalytic activity. However, free hemin is unstable in solution and readily aggregates, reducing catalytic efficiency. We hypothesized that a specific aptamer could bind hemin, stabilize its structure, and enhance its catalytic activity, thereby generating a stronger colorimetric signal suitable for sensitive detection. The sensing mechanism relies on the hemin-aptamer complex catalyzing oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by hydrogen peroxide. Oxidized TMB initially forms a blue product that becomes yellow after addition of acid, allowing quantitative measurement at 450 nm using UV-Vis spectroscopy and qualitative observation by the naked eye. Because aptamers recognize their molecular targets with high specificity, this approach combines selective molecular recognition with simple colorimetric detection. 3. Materials and Methods A hemin-binding DNA aptamer synthesized with high purity was used throughout the study. Reaction conditions were systematically optimized to maximize catalytic performance. Parameters investigated included buffer pH, sodium chloride concentration, magnesium chloride concentration, TMB concentration, hydrogen peroxide concentration, aptamer-hemin incubation time, color-development time, and aptamer stem length. Artificial urine prepared according to a published formulation was employed to simulate real biological samples while maintaining biosafety. Defibrinated sheep blood served as a substitute for human blood during validation experiments. 4. Results and Discussion Initial experiments compared free hemin with the hemin-aptamer complex. UV-Vis spectra showed substantially stronger absorbance at 450 nm when aptamer was present, while visual observation revealed a much darker yellow color. These findings confirmed that aptamer binding significantly enhanced the peroxidase-like activity of hemin. The improvement is attributed to stabilization of the iron center within hemin, promotion of electron transfer between hydrogen peroxide and TMB, and prevention of hemin aggregation. The secondary structure of the aptamer was then optimized. Computational analysis indicated that extending the stem region reduced Gibbs free energy, producing a more stable folded structure. Experimental results demonstrated that increasing the stem length improved catalytic performance up to ten base pairs. Longer stems produced only marginal additional improvement while increasing synthesis cost and the possibility of nonspecific interactions. Consequently, the ten-base-pair stem was selected as the optimal design. Reaction optimization identified pH 5.0 as the best condition because mildly acidic environments favor TMB oxidation while preserving aptamer structure. Sodium chloride concentration of 150 mM maximized signal by stabilizing electrostatic interactions, whereas 50 mM magnesium chloride effectively promoted aptamer folding. TMB concentration reached saturation at 35 mM and hydrogen peroxide at 100 mM, making these conditions both efficient and economical. Five minutes of aptamer-hemin incubation allowed complete complex formation, and approximately 200 seconds of color development generated stable absorbance values. Under optimized conditions, calibration experiments produced a linear analytical range from 0.5 to 7.5 nM hemin. The biosensor achieved a limit of detection of 0.14 nM and a limit of quantification of 0.46 nM. These values are substantially lower than the estimated hemin concentration associated with clinical microhematuria, approximately 2–5 nM, indicating that the sensor possesses sensitivity well beyond the minimum requirement for early screening. Performance was subsequently evaluated in artificial urine. The sensor maintained stable color development despite the presence of urea, creatinine, salts, and other urine components, demonstrating excellent resistance to matrix interference. This result suggests that common urinary constituents do not significantly disrupt aptamer binding or catalytic activity. To further evaluate practical applicability, sheep blood was diluted into artificial urine at concentrations representative of microscopic hematuria. The biosensor successfully detected approximately 0.001% red blood cells, producing visible color changes consistent with spectrophotometric measurements. Performance was comparable to that of commercial urine dipsticks while providing the additional advantage of target-specific molecular recognition through the aptamer. 5. Innovation and Potential Applications The project offers several important innovations. First, it introduces a highly specific hemin-binding aptamer into urine blood detection, improving selectivity compared with conventional oxidation-based strips. Second, systematic optimization of aptamer structure and reaction conditions substantially enhanced analytical performance. Third, the platform employs simple colorimetric chemistry that is inexpensive, rapid, and compatible with point-of-care testing. Finally, the technology provides a promising foundation for domestic production of advanced diagnostic kits in Vietnam. 6. Conclusion Overall, this study successfully demonstrated that a hemin-specific aptamer can significantly enhance hemin catalytic activity and enable highly sensitive detection of microhematuria. The optimized biosensor exhibited strong analytical performance, excellent stability, compatibility with artificial urine, and successful detection of diluted sheep blood. Its analytical sensitivity exceeded current clinical screening requirements, highlighting its potential as a practical diagnostic tool. 7. Future Work Future work will focus on validating the biosensor using clinical urine samples from patients, improving long-term reagent stability, converting the assay into a portable strip format, and optimizing manufacturing procedures for large-scale production. In addition, a smartphone application could be developed to analyze color intensity automatically, reducing subjective interpretation and supporting telehealth-based screening. Together, these developments could facilitate commercialization of a low-cost, highly sensitive, Made-in-Vietnam diagnostic kit capable of expanding access to early urinary disease screening in hospitals, clinics, and community healthcare settings. This platform also demonstrates the broader potential of aptamer-based biosensors for rapid medical diagnostics, as the recognition sequence can be redesigned to detect different biomarkers while maintaining the same simple colorimetric principle.

Organisation: Vinschool Vietnam

Innovator(s): Bui Tu Anh, Hoang Do Uyen Nhi, Hoang Thuy Giang

Category: Medicine, Biotechnology and Medical Devices

Country: Vietnam