Colorimetric Approach Research Articles

Catalytic hairpin assembly-triggered endonuclease-mediated cascade signal recycling for sensitive and colorimetric miRNA detection

AbstractThe catalytic hairpin assembly (CHA)-based miRNA detection methods have garnered significant attention due to their simplicity and acceptable amplification efficiency. However, these methods necessitate improved sensitivity. In this work, we present a colorimetric and ultrasensitive approach for the detection of cancer-related miRNAs which is initiated by CHA-mediated nicking endonuclease-assisted signals recycling. The target initiates the CHA process to expose the functional section in the P2 probe. This section can activate cascaded recycling cycles to produce numerous linker sequences by Nt.AlwI endonuclease-assisted cleavage of two hairpin signal probes. The 3,3’,5,5’-tetramethylbenzidine sulfate (TMB)/H2O2-based color reaction is induced by the fixation of the cDNA-HRP on the surface of magnetic beads, which is mediated by the linker sequence. The proposed method demonstrates a sensitivity that is either comparable to or superior to that of previous colorimetric miRNA detection methods, as a result of this design. Furthermore, the method demonstrated a promising potential for clinical applications and a high selectivity to target miRNA. Consequently, it provides a colorimetric assay that is both ultrasensitive and dependable for the visual detection of miRNA, which has the potential to revolutionize the early diagnosis of cancer.

Analyzing the impact of phosphorous and nitrogen on Castanopsis sclerophylla early growth stages

Plant growth elements, particularly nitrogen (N) and phosphorus (P) are vital for their growth and development, particularly for understory vegetation and their excess limits the net productivity of terrestrial ecosystems. This study focuses on the understory vegetation responds and adaptation to key essential nutrients under changing climate scenarios in subtropical evergreen broad-leaved forests, still needs research attention. this, we set up an experiment taking four treatments in a 50-year-old Castanopsis sclerophylla secondary forest under (a) control (CK), (b) N, (c) P, and (d) combined N and P addition, applied to natural forest regeneration seedlings of C. sclerophylla attained similar growth parameters of diameter of 3 cm and 10 cm height. In addition, carbon, N, P, and non-structural carbohydrates (NSC) were determined through the anthrone colorimetric approach in different parts of seedlings. Results show that the combined N + P application enhanced the N and P by 14.48 %−140.55 % in the seedlings in both dry and wet seasons, respectively. However, during wet season, the content of NSC in the plant leaves significantly exceeded under P addition. Remarkably, CK showed increased P in the growing season but lower during the dry season. Furthermore, the root starch content of seedlings showed a significant increase under the application of N and P compared to combined N + P, ranging between 45.60 % and 58.70 %. Overall, the plant growth is attributed to N and P intake. The nutrient addition and seasonal variations have a coupled effect on seedling growth as proved in the in the natural open forest experiment. The study outcomes emphasize that the alterations in NSC allocation in the roots and leaves of C. sclerophylla seedlings under N + P addition could enhance their adaptation to future global climate changes, drought conditions, and high N concentrations.

Colorimetric approach based on iron oxide magnetic molecularly imprinted nanozyme for sensitive determination of antipyrine and benzocaine: Application to environmental water samples and pharmaceutical dosage form

Nanozymes provide the required enzymatic activity however, they lack the selectivity to target a certain drug during analysis. Herein, a novel polydopamine molecularly imprinted polymer (MIP) was fabricated onto Fe3O4 nanozymes surface for sensitive and selective determination of antipyrine (ANT) and benzocaine HCl (BEN). Synthesized Fe3O4 nanozyme with peroxidase like activity catalyzed the oxidation of ortho phenylenediamine by the aid of H2O2 to produce intense yellow color that can be measured via colorimetric assay. The MIPs were prepared and characterized using field-emission scanning electronic microscopy, Fourier-transform IR spectroscopy, surface area analysis, and X-ray diffraction spectrum. Two Fe3O4 @ MIP NPs nanozyme with peroxidase like activity of two drugs were prepared. Upon binding of the target drug with its respective MIP, an inhibition in the Fe3O4 enzymatic activity occurred and thus a decrease in the yellow color produced. This quantitative decrease in the color intensity represents the drug concentration. Factors affecting the enzymatic colorimetric reaction have been investigated and optimized. The formed MIPs increased the sensitivity of the proposed method with LOD 1.405, 0.658 ng mL−1, LOQ 4.259, 1.993 and recovery percentage of 100.07, 100.57 for ANT and BEN, respectively. The reusability of the Fe3O4 @ MIP NPs was assessed for six cycles without significant loss in enzymatic oxidase activity. Furthermore, the method was validated following ICH guidelines. Then, it was successfully applied for determination of the two drugs in spiked environmental water samples and pharmaceutical formulation.

Advances in gluten detection: A rapid colorimetric approach using core-satellite magnetic particles

Monitoring gluten levels in foods labelled as gluten-free or low-gluten — i.e. containing less than 20 ppm and 100 ppm of gluten, respectively — is crucial for preventing celiac disease-related disorders. Due to their inherent complexity, the standard analytical techniques to assess the gluten content in food are not suitable for on-site measurements, whose need is widely recognized. In this context, we developed a rapid and cost-effective biosensor based on core-satellite magnetic particles (CSMPs) — magnetic cores coated with gold nanoparticles (AuNPs) — further functionalized with anti-gliadin antibodies. The study demonstrates that a 0.016% concentration of the surfactant Tween-20 can induce the spontaneous formation of stable CSMP clusters in dispersion. These clusters, composed of weakly interacting functionalized CSMPs, undergo fragmentation in the presence of gliadin, which specifically binds to the antibodies on the CSMPs. This process results in a colour change, which is measurable by a UV–VIS spectrophotometer. Gliadin extraction was achieved by treating the sample with a non-toxic ethanol-water mixture (60%), sufficient to induce a measurable colour change in the presence of gluten contamination, with a limit of detection (LOD) of 8 ppm, which is lower than low limit established for gluten-free food.

Colorimetric detection of Cu(II) in aqueous solutions using a novel chemosensor: 2-((2-chloroquinolin-3yl)methylidene]amino)phenol

ABSTRACT A spectrophotometric technique based on a selective colorimetric approach is presented in this study for the determination of Cu(II) ions using 2-([(2-chloroquinolin-3-yl)methylidene]amino)phenol (BHMOS) as a detection agent. NMR and FT-IR spectroscopy were used to characterize the compound thoroughly, ensuring accuracy in terms of its molecular structure. As a chemosensor in aqueous solutions, BHMOS has demonstrated a very high level of sensitivity and specificity, even in the presence of other cations. During the interaction between Cu(II) ions and BHMOS, a noticeable colour change was observed to pale green, which is a visual indicator of Cu(II) ions. The method demonstrated linearity for Cu(II) concentrations ranging from 5 to 60 µM, with a detection limit of 1.50 µM. The binding constant for the Cu(II)-BHMOS complex was determined to be 1.14 × 106 M−1, reflecting the strength and stability of the interaction. This method proved to be effective for detecting Cu(II) ions in both natural and simulated water samples, showing its potential utility in environmental monitoring applications.

Enhancing colorimetric efficiency: nanozyme-activated peroxymonosulfate for in situ 3-aminophenol detection.

Anovel colorimetric approach specifically designed to effectively identify the presence of 3-aminophenol (3-AP) in environmental water isintroduced. Briefly, a nitrogen-doped carbon-supported cobalt nanozyme (Co@CN-1) was synthesized and utilized to improve the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of peroxymonosulfate (PMS). Comparative catalytic reactions confirmed that the performance of PMS as an activator exceeds that of hydrogen peroxide catalytically by a factor of 3.5. The catalytic reaction parameters underwent optimization, further resulting in the derivation of a linear detection equation for 3-AP, expressed as inhibition rate (IR%) = 3.35[3-AP]-4.36 (0-20μM, R2 = 0.994) and IR% = 1.43[3-AP] + 31.87 (20-36μM, R2 = 0.992), with the limit of detection (LOD) of 2.84μM. The linear relationship between 3-AP concentration and the conversion of color to grayscale value (GSV) was established by smartphones, expressed as GSV = 1.28[3-AP] + 147.10 (R2 = 0.972). Density functional theory calculations revealed that Co acts as the preferred active site for donating electrons in PMS activation. This work provides a rapid and accurate approach for monitoring 3-AP concentration, enabling real-time analysis and potentially contributing to environmental and ecological studies.

Unveiling the potential of the capillary-driven microfluidic paper-based device integrated with smartphone-based for simultaneously colorimetric salivary ethanol and △9-tetrahydrocannabinol analysis

Monitoring various biomarkers in saliva samples emerges as a dynamic and non-invasive method. However, the high viscosity of saliva presents a distinct challenge when integrating paper-based platforms for on-site analysis. In addressing this challenge, we introduced the capillary-driven microfluidic paper-based analytical devices (μCD-PAD) designed for user-friendly and simultaneous detection of ethanol and tetrahydrocannabinol (THC) in saliva without a sample preparation step. Employing a colorimetric approach, we quantified both analytes. Synthetic salivas of varying viscosity flowed seamlessly to the detection zone without needing a sample preparation step, and no impact on colorimetric detection due to viscosity was observed (RSD <5 %). Within 10 min after the solution reached the detection zone, the device produced a homogeneous color signal, easily analyzed by a smartphone camera. To extend the application for determination to cover a legal limit concentration of ethanol and concentration of salivary THC even 24 h after marijuana consumption, the detection time of 30 min was optimized. Moreover, a saliva sample containing both analytes was used to demonstrate the capability of the developed device to detect ethanol and THC simultaneously. No cross-talk between ethanol and THC occurred and showed recovery in the 98–102 % for ethanol and 95–105 % for THC with acceptable accuracy. This developed device exhibits excellent potential for forensic applications, providing a user-friendly, cost-effective, and real-time screening tool for detecting ethanol and THC in saliva.

Identification of Pristine and Protein Corona Coated Micro- and Nanoplastic Particles with a Colorimetric Sensor Array.

A colorimetric sensor array has been developed to differentiate various micro- and nanoplastic particles (MNPs), both pristine and those coated with a protein corona, in buffered water. This array utilizes five distinct cross-reactive chemo-responsive dyes, which exhibit changes in visible optical absorbance upon interaction with MNPs. Although no single dye responds exclusively to either pristine or protein-corona-coated MNPs, the collective shifts in color across all dyes create a unique molecular fingerprint for each type of MNP. This method demonstrates high sensitivity, capable of detecting MNPs of various sizes (50 nm, 100 nm, and 2 μm) and differentiating them from controls at concentrations as low as 10 ng/mL using standard chemometric techniques, ensuring accurate results without error. Additionally, the method can effectively distinguish between pristine and protein-corona-coated polystyrene MNPs. This colorimetric approach offers a rapid, cost-effective, and accurate method for monitoring MNP pollution and assessing their prior interactions with biological systems.

Intrinsic oxidase-mimicking activity of nitrite upon visible light illumination and its colorimetric detection in saliva

Small molecules with enzyme-like properties have recently attracted considerable attention. Herein, we discovered that nitrite possesses intrinsic oxidase-mimicking activity upon visible light, catalyzing the oxidation of the typical chromogenic substrate in the absence of H2O2. Notably, nitrite exhibited a markedly high value of Kcat, approximately 4, 7, and 4000-fold greater than that of Acr+-Mes, Eosin Y, and Diacetyl, respectively. Comprehensive investigation elucidated that O2•⁻ and •OH are the primary reactive oxygen species (ROS) responsible for the oxidation of 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate (TMB). Leveraging the linear correlation between the absorbance of oxidized TMB (oxTMB) at 652 nm and nitrite concentration, a simple colorimetric approach for nitrite detection was successfully established in the range of 1–75 μM with a detection limit of 0.14 μM. Moreover, the proposed strategy could be applied to determine the nitrite concentration in saliva, exhibiting a great prospect for clinical diagnosis. This work contributes novel insights into the exploration of small-molecule enzyme mimics.

Sesame Detection in Food Using DNA-Functionalized Gold Nanoparticles: A Sensitive, Rapid, and Cost-Effective Colorimetric Approach.

Food safety control is a key issue in the food and agriculture industries. For such purposes, developing miniaturized analytical methods is critical for enabling the rapid and sensitive detection of food supplements, allergens, and pollutants. Here, a novel bioanalytical methodology based on DNA-functionalized gold nanoparticles (AuNPs) and colorimetric detection was developed to detect the presence of sesame (a major allergen) through sesame seed DNA as a target, in food samples. The presence of sesame DNA induces controlled nanoparticle aggregation/desegregation, resulting in a color change (from blue to red) proportional to sesame DNA concentration. The incorporation of multicomponent nucleic acid enzymes (MNAzymes) in this strategy has been carried out to perform an isothermal signal amplification strategy to improve the sensitivity of detection. Also, open-source software for color analysis was used to ensure an unbiased visual color-change detection, enhancing detection accuracy and sensitivity and opening the possibility of performing a simple and decentralized analyte detection. The method successfully detected the presence of sesame DNA in sesame seed, sesame oil, olive oil, and sunflower oil. In brief, the developed approach constitutes a simple and affordable alternative to perform a highly sensitive detection of DNA in food without complex methodologies or the requirement of expensive instrumentation.

Visual arsenic detection in environmental waters: Innovating with a naked-eye biosensor for universal application

Arsenic contamination in environmental water sources poses a significant threat to human health, necessitating the development of sensitive and accessible detection methods. This study presents a multidimensional optimization of a bacterial biosensor for the susceptible and deoxyviolacein (DV)-based visual detection of arsenic. The research involved screening six different arsenic resistance (ars) operons and optimizing the genetic circuit to minimize background noise. Introducing an arsenic-specific transport channel enhanced the sensor's sensitivity to 1 nM with a quantitative range from 0.036 to 1.171 μM. The pigment-based biosensor offers a simple colorimetric approach for arsenic detection without complex instrumentation. The preferred biosensor demonstrated characteristics of anti-chelating agent interference, consistently quantified As(III) concentrations ranging from 0.036 to 1.171 μM covering the World Health Organization (WHO) drinking water limit. Innovatively, it effectively detects arsenic in seawater within a linear regression range of 0.071 to 1.125 μM. The biosensor's selectivity for arsenic was confirmed, with minimal cross-response to group 15 metals. Our naked-eye biosensor offers a novel approach for the rapid, on-site detection of arsenic in various water sources. Its simplicity, cost-effectiveness, and versatility make it a valuable tool for environmental monitoring and public health initiatives.

Low-cost, immediate, general-purpose, and high-throughput (LIGHt) smartphone colorimetric screening assay for water-soluble protein

An efficient and rapid method for the detection of total soluble protein in tobacco leaves, utilizing a smartphone-based colorimetric approach has been developed. The proposed low-cost, immediate, general-purpose, and high-throughput (LIGHt) smartphone colorimetric screening assay integrates commercially available microplates, enabling on-site, high-throughput screening of tobacco leaf quality. The study involves preparing protein standard solutions and constructing standard curves using both spectrophotometric and smartphone-based methods. The LIGHt smartphone colorimetry yielded an average relative standard deviation of 10.6 %, a limit of detection of 2 μg/mL, and an average recovery of 93 %. The results demonstrated a comparable performance between intensities from the blue channel and the absorbance values in reflecting protein concentrations, validating the feasibility of utilizing smartphone colorimetry for protein concentration determination. Our approach demonstrates the potential for practical implementation in the field, providing a cost-effective and user-friendly solution for rapid quality assessment in the tobacco industry. The LIGHt smartphone colorimetry enhances quality control practices in the tobacco sector and offers a promising tool for on-site production quality testing in various industries, such as fruits and vegetables.

Colorimetric detection of sitagliptin and moxifloxacin using AgNPs modified by 4-mercaptophenylbronic acid

Nowadays, considering the vital role of drugs, identifying and diagnosing them with simple and cost-effective methods and materials is very important. This study explores a colorimetric approach using silver nanoparticles (AgNPs) modified with 4-mercaptophenylboronic acid (MPBA) as a rapid and economical sensor for detecting Sitagliptin (Sit) and Moxifloxacin (Moxi) drugs. The MPBA-AgNPs sensor was synthesized via chemical synthesis of AgNPs followed by surface modification by MPBA. The effect of different parameters on the responsiveness of the MPBA-AgNPs sensor was investigated. The surface morphology, chemical structure, and size of the MPBA-AgNPs sensor in different conditions were examined by FE-SEM, FT-IR, DLS, and TEM analysis. Finally, the results of various investigations showed that the surface of AgNPs successfully modified by MPBA and MPBA-AgNPs as a colorimetric sensor for the detection of Sit and Moxi drugs has excellent detection limits of 16.63 and 0.27 µM. The recovery of the experiment in real samples was obtained in the range of 92.37–105.44 %.

Colorimetric sensor nanoarchitectonics for trace Pb2+ ions detection by porous fungus-like CoS nanozyme

The heavy metal lead (Pb) holds significant importance in the manufacturing industry due to its extensive range of applications. However, its high toxicity poses a serious risk to both environment and human health. Consequently, on-site portable analysis methods have been developed to detect Pb2+, which is advantageous for obtaining timely information regarding environmental pollution. In this study, a fungal-like porous CoS (FP CoS) was discovered to selectively recognize, adsorb, and detect Pb2+. FP CoS exhibited rapid kinetics (reducing from 8.56 mg∙L–1 to 69 µg∙L–1 within 30 min) as well as exceptional adsorption capacity of 88.14 mg∙g−1 for Pb2+. Benefiting from the selective enrichment of Pb2+, the peroxidase-like activity of FP CoS nanoenzyme was further enhanced, enabling colorimetric detection of Pb2+ with a detection limit of 14 ng∙L–1, surpassing numerous reported materials. When applied to environmental samples spiked with 0–100 μg∙L–1 Pb2+, the combination of FP CoS nanosensor and a portable RGB color sensor yielded an impressive recovery percentage of 112 %. Based on these results, the FP CoS nanosensor not only provides a rapid and visually discernible colorimetric approach for trace and ultra-trace Pb2+ but also serves as a sensitive sensor for detecting Pb2+ in various scenarios.

Development of an organophosphate and carbamate pesticide test kit by using a magnetic particle coated with esterase enzyme from honey bee heads

Organophosphates (OPs) and carbamates (CMs) play a vital role in Thai agriculture, public health, and pest control. However, their detection presents challenges due to expensive methods requiring specialized expertise. Addressing this gap, our study introduces an innovative, cost-effective method for detecting OPs and CMs in Thailand. We utilized the test kit by using magnetic particles coated with esterase enzymes from honey bee heads combined with a colorimetric approach. The developed test kit shows low limits of detection (LODs) at 0.001 mg/L of dichlorvos (OPs) and 0.004 mg/L for carbaryl (CMs). The efficiency of the developed test kits in comparison with the standard technique of gas chromatography with flame photometry detection (GC-FPD) shows a comparable result in fruit and vegetable residue analysis. This developed test kit proposes a cost-effective OPs and CMs analysis test kit for Thailand, promising expense reduction and simplified verification processes. Its applicability to fruits and vegetables signifies practicality in agriculture, marking a significant advancement in accessible and efficient pesticide residue monitoring. Despite challenges in pesticide use, our method holds promise for improving safety and efficiency in Thai pesticide management.Graphical

Development of a paper-based transcription aptasensor for convenient urinary uric acid self-testing

The abnormal uric acid (UA) level in urine can serve as warning signals of many diseases, such as gout and metabolic cardiovascular diseases. The current methods for detecting UA face limitations of instrument dependence and the requirement for non-invasiveness, making it challenging to fulfill the need for home-based application. In this study, we designed an aptasensor that combined UA-specific transcriptional regulation and a fluorescent RNA aptamer for convenient urinary UA testing. The concentration of UA can be translated into the intensity of fluorescent signals. The aptasensor showed higher sensitivity and more robust anti-interference performance. UA levels in the urine of different volunteers could be accurately tested using this method. In addition, a paper-based aptasensor for UA self-testing was manufactured, in which the urinary UA levels could be determined using a smartphone-based colorimetric approach. This work not only demonstrates a new approach for the design of disease-associated aptasensor, but also offers promising ideas for home-based detection of UA.

Immobilization of a broad host range phage on the peroxidase-like Fe-MOF for colorimetric determination of multiple Salmonella enterica strains in food.

Abroad host range phage-based nanozyme (Fe-MOF@SalmpYZU47) was prepared for colorimetric detection of multiple Salmonella enterica strains. The isolation of a broad host range phage (SalmpYZU47) capable of infecting multiple S. enterica strains was achieved. Then, it was directly immobilized onto the Fe-MOF to prepare Fe-MOF@SalmpYZU47, exhibiting peroxidase-like activity. The peroxidase-like activity can be specifically inhibited by multiple S. enterica strains, benefiting from the broad host range capture ability of Fe-MOF@SalmpYZU47. Based on it, a colorimetric detection approach was developed for S. enterica in the range from 1.0 × 102 to 1.0 × 108CFUmL-1, achieving a low limit of detection (LOD) of11CFUmL-1. TheFe-MOF@SalmpYZU47 was utilized for detecting S. enterica in authentic food samples, achieving recoveries ranging from 91.88 to 105.34%. Hence, our proposed broad host range phage-based nanozyme exhibits significant potential for application in the colorimetric detection of pathogenic bacteria.

Gold nanoparticles assisted colorimetric sensing of paroxetine, duloxetine, and olanzapine in aqueous and micellar systems with improved sensitivity

The present study explored a rapid and facile colorimetric identification of two antidepressants (paroxetine and duloxetine) and one antipsychotic drug (olanzapine) in aqueous and micellar systems using citrate-stabilized gold nanoparticles (Cit-AuNPs). The sensing approach involved hydrogen bonding and electrostatic interactions on the surface of Cit-AuNPs. The attributes of Cit-AuNPs and their interactions with the drugs were probed using characterization techniques like UV–Vis spectroscopy, scanning electron microscopy (SEM), dynamic light scattering (DLS), zeta potential measurement, and Fourier-transform infrared spectroscopy (FT-IR). Various factors including the volume of Cit-AuNPs, pH, reaction time, surfactant concentration, and the sequence of addition of reagents were optimized to inspect their impacts on sensing results. Under optimized conditions, the probe showed good linearity for the three drugs at the nanomolar (nM) range. Consequently, the color of the Cit-AuNP solution transformed from red to royal purple and blue, which was visible to the naked eye and monitored by visible absorption spectra. This label-free optical detection approach could recognize paroxetine (Prx), duloxetine (Dlx), and olanzapine (Olz) in aqueous system at concentrations ranging from 1 to 17 nM with a limit of detection (LOD) of 0.312, 0.258, and 0.418 nM (S/N = 3), respectively. Meanwhile, the sensitivity was improved in the Triton X-100 (TX-100) micellar system with LOD of 0.029, 0.030, and 0.029 nM for Prx, Dlx, and Olz, respectively accompanied by a shift in the absorption bands. The micellar system contributes stability to the Cit-AuNP-drug complex. The devised colorimetric approach was convenient and did not require any complex equipment. It was efficaciously used to detect three drugs in pharmaceutical products, spiked human urine, and blood serum samples with satisfactory recoveries of 99.83–103.45% and RSD < 3.5%. Notably, the sensing assay demonstrated good selectivity for three drugs in the existence of commonly encountered foreign species and certain common drugs with a relative error below 3%. The results of the present study were statistically compared with reference methodology. The results were validated with no significant difference between the devised and reported approaches considering precision and accuracy, respectively.

Turn-off enzyme activity of histidine-rich peptides for the detection of lysozyme.

Anassay that integrateshistidine-rich peptides (HisRPs) with high-affinity aptamerswas developed enabling the specific and sensitive determination of the target lysozyme. The enzyme-like activity of HisRP is inhibited by its interaction with a target recognized by an aptamer. In the presence of the target, lysozyme molecules progressively assemble on the surface of HisRP in a concentration-dependent manner, resulting in the gradual suppression of enzyme-like activity. This inhibition of HisRP's enzyme-like activity can be visually observed through color changes in the reaction product or quantified using UV-visible absorption spectroscopy. Under optimal conditions, the proposed colorimetric assay for lysozyme had a detection limit as low as 1 nM and exhibited excellent selectivity against other nonspecific interferents. Furthermore, subsequent research validated the practical applicability of the developed colorimetric approach to saliva samples, indicating that the assay holds significant potential for the detection of lysozymes in samples derived from humans.

Antiurolithiatic activity of Indian medicinal plant: Ocimum kilimandscharicum Gurke (Lamiaceae)

Urolithiasis is the most prevalent condition of the urinary system, characterized by the formation of stones inside the urinary tract. It is urgent to look for a natural urolithiasis therapy due to the serious side effects of conventional medications. Hydro-alcoholic (80% v/v) extract of the aerial parts of Ocimum kilimandscharicum (OK) and its ethyl acetate, chloroform, n-butanol, aqueous, and n-hexane fractions were subjected to in vitro antiurolithiatic screening as well as preliminary screening of phytochemicals. The in vitro antiurolithiatic activity of O. kilimandscharicum was studied using its hydroalcoholic extract (HAEOK). Calcium phosphate test using a colorimetric approach and calcium oxalate assay using a titrimetric model were used to determine the proportion of calcium oxalate crystals that dissolved. Total phenolic content (TPC) and total flavonoid content (TFC) were measured for the extract and fractions of OK. Ethyl acetate fraction (EAFOK) had a greater capacity to suppress crystal formation in both the calcium phosphate and calcium oxalate assays. The percent dissolution of calcium oxalate by HAEOK and EAFOK (31.48 ± 0.920% and 39.21 ± 0.903%) and calcium phosphate crystals by HAEOK and EAFOK (59.03 ± 0.820% and 66.62 ± 0.468%) was determined, respectively. At p &lt; 0.05 and p &lt; 0.01, differences between the results were regarded as significant. Cystone was employed as a standard drug. This study revealed that EAFOK showed significant antiurolithiatic activity. The antiurolithiatic activity of the extract/fraction was attributed to the steroids, triterpenoids, and flavonoid content of OK.