Online preconcentration and determination of chondroitin sulfate, dermatan sulfate and hyaluronic acid in biological and cosmetic samples using capillary electrophoresis.

Capillary electrophoresis with large-volume sample stacking using an electroosmotic flow pump was developed for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid. Central composite design was used to simultaneously optimize the parameters for capillary electrophoresis separation. The optimized capillary electrophoresis conditions were 200 mM sodium dihydrogen phosphate, 200 mM butylamine, and 0.5% w/v polyethylene glycol as a background electrolyte, pH 4 and -16 kV. Exploiting large-volume sample stacking using an electroosmotic flow pump, the sensitivity of the proposed capillary electrophoresis system coupled with UV detection was significantly improved with limits of detection of 3, 5, 1 mg/L for chondroitin sulfate, dermatan sulfate, and hyaluronic acid, respectively. The developed method was applied to the determination of chondroitin sulfate and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic products, and supplementary samples with highly acceptable accuracy and precision. Therefore, the proposed capillary electrophoresis approach was found to be simple, rapid, and reliable for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic, and supplementary samples without sample pretreatment.

Highly Sensitive Detection of Salmonella typhimurium Using a Colorimetric Paper-Based Analytical Device Coupled with Immunomagnetic Separation.

Salmonella causes over a million foodborne illnesses per year in the United States resulting in more hospitalizations and deaths than any other foodborne bacterial pathogen. To help prevent outbreaks, a rapid, portable, sensitive, and reliable method for onsite detection of bacteria that can be used in different sample matrices would be beneficial. Herein, we present a colorimetric paper-based analytical device (PAD) combined with immunomagnetic separation (IMS) for detecting Salmonella typhimurium. IMS anti-Salmonella coated magnetic beads were applied to capture and separate bacteria from the sample matrix and preconcentrate it into small volumes before testing on paper. To directly detect S. typhimurium after IMS, a sandwich immunoassay was implemented into the procedure with ß-galactosidase (ß-gal) as the detection enzyme. Using the antibody/enzyme complex, we performed a colorimetric assay with chlorophenol red-ß-d-galactopyranoside (CPRG) for bacteria quantification. The method was confirmed to be highly specific to S. typhimurium without interference from other pathogenic bacteria like Escherichia coli. Using this system, the limit of detection of S. typhimurium was found to be 102 CFU mL-1 in culturing solution without any pre-enrichment. In addition, distance-based detection where the concentration is read as the length of colored band formed on the reaction was also demonstrated. This assay had a detection limit of 102 CFU mL-1 for S. typhimurium, providing an instrument-free quantitative analysis alternative to spot tests, which require image analysis. Finally, the proposed platform was applied for detection of S. typhimurium in inoculated Starling bird fecal samples and whole milk with detection limits of 105 CFU g-1 and 103 CFU mL-1, respectively, and this is the first published paper-based detection method for S. typhimurium in bird feces and whole milk.

A rapid and facile immunoassay for C-reactive protein using PDMS-based digital magnetofluidics.

BACKGROUND: C-reactive protein has been reported as a biomarker of inflammation caused by acute injury, infection or tissue damage and also a prediction marker of cardiovascular diseases. Commonly, the gold standard for the detection of CRP is enzyme-linked immunosorbent assays (ELISAs). Normally, traditional immunoassays in multiwell plates typically suffer from prolonged assay time due to slow mass transport controlled by diffusion. Herein, a PDMS based magnetofluidic approach has been applied for a rapid and facile immunoassay using a sandwich enzyme-linked immunosorbent assay (ELISA) for the analysis of CRP. RESULTS: Due to the superhydrophobic PDMS, droplets of reagent and sample solutions were obtained when pipetting all solutions onto the PDMS substrate. These droplets were individually controlled by an external magnet to perform the assays. Magnetic beads immobilized with a capture antibody were not only used for immunomagnetic separation (IMS) of the captured CRP from the sample matrix, but also used as a carrier for droplet movement on the magnetofluidic device, expediting the immunoassay procedure, especially washing steps. The immunoassay of CRP was successfully performed within 1 h with a limit of detection of 0.015 mg L-1 in the concentration range of 0.1-10 mg L-1. The recovery percentages of CRP spiked in human serum were found in the range of 90-114 % with %RSD of less than 5 %, indicating acceptable accuracy and precision. SIGNIFICANCE: By individually controlling the droplet movement using an external magnet, all steps of immunoassays were simply and rapidly performed. In addition, the microfluidic format allows for small volumes of reagents and samples and rapid assay kinetics. Therefore, the proposed magnetofluidic approach has shown its potential of becoming a rapid, facile and cost-effective method to perform traditional immunoassays in a variety of applications. In addition, the proposed approach is also particularly well-suited for analyses/reactions with multiple steps.

Unraveling the role of gut microbiota by fecal microbiota transplantation in rat model of kidney stone disease.

Emerging research on the microbiome highlights the significant role of gut health in the development of kidney stones, indicating that an imbalance in gut bacteria or dysbiosis can influence the formation of stones by altering oxalate metabolism and urinary metabolite profiles. In particular, the overabundance of specific bacteria such as Enterococcus and Oxalobacter spp., which are known to affect oxalate absorption, is observed in patients with urolithiasis. This study investigates the effects of gut dysbiosis on urolithiasis through fecal microbiota transplantation (FMT) from patients to rats and its impact on urinary mineral excretion and stone formation. Fecal samples from eight patients with calcium oxalate stones and ten healthy volunteers were collected to assess the gut microbiome. These samples were then transplanted to antibiotic-pretreated Wistar rats for a duration of four weeks. After transplantation, we evaluated changes in the fecal gut microbiome profile, urinary mineral excretion rates, and expression levels of intestinal zonula occluden-1 (ZO-1), SLC26A6 and renal NF-κB. In humans, patients with urolithiasis exhibited increased urinary calcium and oxalate levels, along with decreased citrate excretion and increased urinary supersaturation index. The fecal microbiota showed a notable abundance of Bacteroidota. In rodents, urolithiasis-FMT rats showed urinary disturbances similar to patients, including elevated pH, oxalate level, and supersaturation index, despite negative renal pathology. In addition, a slight elevation in the expression of renal NF-κB, a significant intestinal SLC26A6, and a reduction in ZO-1 expression were observed. The gut microbiome of urolithiasis-FMT rats showed an increased abundance of Bacteroidota, particularly Muribaculaceae, compared to their healthy FMT counterparts. In conclusion, the consistent overabundance of Bacteroidota in both urolithiasis patients and urolithiasis-FMT rats is related to altered intestinal barrier function, hyperoxaluria, and renal inflammation. These findings suggest that gut dysbiosis, characterized by an overgrowth of Bacteroidota, plays a crucial role in the pathogenesis of calcium oxalate urolithiasis, underscoring the potential of targeting the gut microbiota as a therapeutic strategy.

Separation selectivity patterns of fully charged achiral compounds in capillary electrophoresis with a neutral cyclodextrin.

Based on the separation selectivity equation, related to the dimensionless parameters for fully charged achiral analytes using a neutral CD, the separation selectivity can be classified into seven patterns. With respect to CZE without CD, the presence of CD in the buffer may improve, or reduce, the separation selectivity with this effect being accompanied by the same or reversed electrophoretic mobility order for charged analytes. This can depend on the separation selectivity of the two analytes in free solution, the binding selectivity, the separation selectivity of analyte-CD complexes and the ratio of electrophoretic mobility of the analytes in free, and complexed forms. Using positional isomers of benzoic acids and phenoxy acids as test analytes and α-CD as a selector, the observed separation selectivity shapes were found to be in excellent agreement with the predicted separation selectivities.

Simultaneous colorimetric detection of nephrolithiasis biomarkers using a microfluidic paper-based analytical device.

A microfluidic paper-based analytical device (µPAD) coupled with colorimetric detection was developed for simultaneous determination of urinary oxalate, citrate and uric acid (UA) which are important biomarkers of nephrolithiasis or kidney stones. The colorimetric detections were based on enzymatic reactions using oxalate oxidase and uricase for oxalate and UA, respectively, while an indicator displacement assay (IDA) using a copper murexide complex was applied for citrate detection. The developed µPAD was successfully applied for simultaneous determination of the three biomarkers in urine within 25 min, with linear ranges of 2-40, 5-150, and 5-45 mg L-1 and detection limits of 0.6, 2.9 and 3.1 mg L-1 for oxalate, UA, and citrate, respectively. The values of the percent relative standard deviation (% RSD) were lower than 6.4% for inter-day and intraday measurements of oxalate, citrate and UA standards spiked in urine samples with recovery percentages in the range of 81.0-109.8%, indicating acceptable accuracy and precision of the developed method for determination of the three biomarkers in urine samples. Accordingly, the developed µPAD holds great promise to be a simple, fast, inexpensive, low-sample and reagent volume, reliable and portable tool for simultaneous determination of oxalate, citrate and UA in urine, especially for on-site analysis.

Premature Senescence and Telomere Shortening Induced by Oxidative Stress From Oxalate, Calcium Oxalate Monohydrate, and Urine From Patients With Calcium Oxalate Nephrolithiasis.

Oxidative stress, a well-known cause of stress-induced premature senescence (SIPS), is increased in patients with calcium oxalate (CaOx) kidney stones (KS). Oxalate and calcium oxalate monohydrate (COM) induce oxidative stress in renal tubular cells, but to our knowledge, their effect on SIPS has not yet been examined. Here, we examined whether oxalate, COM, or urine from patients with CaOx KS could induce SIPS and telomere shortening in human kidney (HK)-2 cells, a proximal tubular renal cell line. Urine from age- and sex-matched individuals without stones was used as a control. In sublethal amounts, H2O2, oxalate, COM, and urine from those with KS evoked oxidative stress in HK-2 cells, indicated by increased protein carbonyl content and decreased total antioxidant capacity, but urine from those without stones did not. The proportion of senescent HK-2 cells, as indicated by SA-ßgal staining, increased after treatment with H2O2, oxalate, COM, and urine from those with KS. Expression of p16 was higher in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than it was in cells treated with urine from those without stones and untreated controls. p16 was upregulated in the SA-ßgal positive cells. Relative telomere length was shorter in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than that in cells treated with urine from those without stones and untreated controls. Transcript expression of shelterin components (TRF1, TRF2 and POT1) was decreased in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS, in which case the expression was highest. Urine from those without KS did not significantly alter TRF1, TRF2, and POT1 mRNA expression in HK-2 cells relative to untreated controls. In conclusion, oxalate, COM, and urine from patients with CaOx KS induced SIPS and telomere shortening in renal tubular cells. SIPS induced by a lithogenic milieu may result from upregulation of p16 and downregulation of shelterin components, specifically POT1, and might contribute, at least in part, to the development of CaOx KS.

Mapping of fluidic mixing in microdroplets with 1 micros time resolution using fluorescence lifetime imaging.

Microdroplets generated in microfluidic channels hold great promise for use as substrates in high-throughput chemical and biological analysis. These water-in-oil compartments can serve as isolated reaction vessels, and since they can be generated at rates in excess of 1 kHz, thousands of assays can be carried out quickly and reproducibly. Nevertheless, sampling the large amount of information generated from these platforms still remains a significant challenge. For example, considering the high droplet generation rates and velocities, reproducibility and micrometer resolution are challenging requirements that must be fulfilled. Herein we combine confocal fluorescence lifetime imaging microscopy with a statistical implementation that permits the analysis of mixing phenomena within microdroplets with a temporal resolution of 1 mus. Importantly, such exquisite resolution is only possible as a result of the large number of droplets sampled and their high structural reproducibility.

Clinical validation of urinary indole-reacted calcium oxalate crystallization index (iCOCI) test for diagnosing calcium oxalate urolithiasis.

An indole-reacted calcium oxalate crystallization index (iCOCI) test was developed to quantify the total competence of urine to precipitate calcium oxalate (CaOx) crystals. We conducted the prospective cohort study in accordance with the STARD guideline to evaluate the accuracy of urinary iCOCI test (index test) for diagnosing urolithiasis. A total of 281 participants were recruited for the study. Levels of urinary iCOCI were determined in the pre-diagnostic 24-h urine samples. Positive urinary iCOCI (≥ 0.6 COM eqv., g/L) was accounted for 51% (144/281), and the rest of 49% (137/281) were negative. Non-contrast CT imaging (reference standard) was subsequently performed for the definite diagnosis of urolithiasis to divide the participants into two groups, non-stone subjects (NSS, n = 122) and stone-forming subjects (SFS, n = 159). It should be noted that only subjects who currently had urinary stone at the time of study were classified as SFS. Urinary iCOCI levels in the SFS were significantly higher than the NSS. ROC analysis revealed an area under curve (AUC) of 0.893 (95% CI: 0.855-0.932) in separating NSS from all SFS. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, positive likelihood ratio (LH+) and negative likelihood ratio (LH-) of urinary iCOCI test for diagnosis of all urolithiasis were 87%, 80%, 84%, 84%, 83%, 4.44 and 0.16, respectively. Of 159 SFS, 38 were confirmed to have CaOx stones. Among these 38 CaOx SFS, only 2 had negative urinary iCOCI test. The AUC of urinary iCOCI test for separating CaOx SFS from NSS was markedly high (0.946, 95% CI: 0.914-0.978). Sensitivity, specificity, PPV, NPV, accuracy, LH+ and LH- of urinary iCOCI test for diagnosing CaOx urolithiasis were 95%, 86%, 68%, 98%, 88%, 6.80 and 0.06, respectively. Conclusion, we clinically validated that an innovative non-invasive urinary iCOCI test was highly accurate to diagnose urolithiasis, especially CaOx stone. With its high sensitivity and NPV, urinary iCOCI test is clinically intended to use as a screening test for CaOx urolithiasis. LH- of 0.06 indicates that negative result of urinary iCOCI test is highly accurate to rule out the CaOx stone formation. It is noted that urinary iCOCI level is expressed as arbitrary unit, and it is not directly related to the actual physiological level of urinary oxalate.

Analysis of protein-protein interactions by using droplet-based microfluidics.

Every little drop: The K(D) values of angiogenin (ANG) interactions as shown by FRET analysis of thousands of pL-sized droplets agree with data from bulk-fluorescence polarization measurements. Importantly, the use of fluorophores does not affect the activity of ANG or the binding of anti-ANG antibodies to ANG. Such an experimental platform could be applied to the high-throughput analysis of protein-protein interactions.

Identification of rare progenitor cells from human periosteal tissue using droplet microfluidics.

The isolation and characterisation of single cells from a heterogeneous population are important processes in cell biology, immunology, stem cell research, and cancer research. In the development of novel cell-based therapies, there is a considerable need to target specific cell types to allow for further analysis and amplification ex vivo. We introduce, herein, the use of droplet-based microfluidics as a platform technology for the identification and quantification of distinct cell phenotypes. Using molecular labelling of specific cell populations by antibodies and fluorescent dyes, detection of single cells encapsulated within picolitre-sized aqueous droplets can be performed using high-sensitivity confocal fluorescence detection. Specifically, rare progenitor cells were immunodetected within a heterogeneous population of cells isolated from human periosteal tissue. Using this model human cell population, the accuracy and reproducibility of the droplet system were tested and the results were verified using conventional flow cytometry. It was found that the quantitation of phenotypic subpopulations measured using both techniques is directly comparable. Accordingly, this study demonstrates the biological capacity of droplet-based microfluidics for cellular analysis and provides a necessary first step towards the development of a novel cell sorting technology.

Droplet microfluidics: from proof-of-concept to real-world utility?

Droplet microfluidics constitutes a diverse and practical tool set that enables chemical and biological experiments to be performed at high speed and with enhanced efficiency when compared to conventional instrumentation. Indeed, in recent years, droplet-based microfluidic tools have been used to excellent effect in a range of applications, including materials synthesis, single cell analysis, RNA sequencing, small molecule screening, in vitro diagnostics and tissue engineering. Our 2011 Chemical Communications Highlight Article [Chem. Commun., 2011, 47, 1936-1942] reviewed some of the most important technological developments and applications of droplet microfluidics, and identified key challenges that needed to be addressed in the short term. In the current contribution, we consider the intervening eight years, and assess the contributions that droplet-based microfluidics has made to experimental science in its broadest sense.

Microdroplets: a sea of applications?

The exploitation of microdroplets produced within microfluidic environments has recently emerged as a new and exciting technological platform for applications within the chemical and biological sciences. Interest in microfluidic systems has been stimulated by a range of fundamental features that accompany system miniaturization. Such features include the ability to process and handle small volumes of fluid, improved analytical performance when compared to macroscale analogues, reduced instrumental footprints, low unit cost, facile integration of functional components and the exploitation of atypical fluid dynamics to control molecules in both time and space. Moreover, microfluidic systems that generate and utilize a stream of sub-nanolitre droplets dispersed within an immiscible continuous phase have the added advantage of allowing ultra-high throughput experimentation and being able to mimic conditions similar to that of a single cell (in terms of volume, pH, and salt concentration) thereby compartmentalizing biological and chemical reactions. This review provides an overview of methods for generating, controlling and manipulating droplets. Furthermore, we discuss key fields of use in which such systems may make a significant impact, with particular emphasis on novel applications in the biological and physical sciences.

Monitoring of real-time streptavidin-biotin binding kinetics using droplet microfluidics.

Rapid kinetic measurements are important in understanding chemical interactions especially for biological molecules. Herein, we present a droplet-based microfluidic platform to study streptavidin-biotin binding kinetics with millisecond time resolution. With integration of a confocal fluorescence detection system, individual droplets can be monitored and characterized online to extract kinetic information. Using this approach, binding kinetics between streptavidin and biotin were observed via fluorescence resonance energy transfer. The binding rate constant of streptavidin and biotin was found to be in a range of 3.0 x 10 (6)-4.5 x 10 (7) M (-1) s (-1).

IR-Compatible PDMS microfluidic devices for monitoring of enzyme kinetics.

Coupling infrared (IR) spectroscopy to microfluidic devices provides a powerful tool for characterizing complex chemical and biochemical reactions. Examples of microfluidic devices coupled with infrared spectroscopy have been limited, however, largely due to the difficulties associated with fabricating systems in common infrared transparent materials like CaF2. Recent reports have shown that polydimethylsiloxane (PDMS) can be used as an IR transparent substrate when fabricated with thin layers. The use of soft lithography with PDMS expands the library of possible designs that can be achieved for IR measurements in microfluidics. In initial reports with thin PDMS, the target analytes were small molecules; however, IR spectroscopy offers a powerful tool to study protein structure and reactions. Here, a PDMS microfluidic device compatible with IR spectroscopy was fabricated by means of spin-coating of PDMS pre-polymer to obtain thin PDMS microfluidic features. The device was comprised of only PDMS and IR absorption of PDMS was significantly minimized due to the thickness (∼40 µm) of the PDMS layer. The use of thin PDMS allowed for measuring the amide I and II vibrational bands of proteins that have been difficult to measure in other microfluidic devices. To demonstrate the power of the system, the microfluidic device was successfully used to measure the enzyme kinetics as one class of important biochemical reactions with broad use in a variety of fields from medicine to biotechnology. As a model, the reaction of glucose oxidase with glucose was tracked by following the formation of gluconic acid. Michaelis-Menten kinetics from the device were compared with bulk solution measurements and found to be in good agreement.

Droplet-based glucosamine sensor using gold nanoparticles and polyaniline-modified electrode.

A droplet-based electrochemical sensor for direct measurement of D-glucosamine was developed using carbon paste electrodes (CPEs) modified with gold nanoparticles (AuNPs) and polyaniline (PANI). Central composition design (CCD) was employed as a powerful method for optimization of parameters for electrode fabrication. The optimized amounts of AuNPs and PANI obtained from the response surface were determined as 300 and 3000mgL(-1), respectively. Coupled with a droplet microfluidic system, the analysis of glucosamine was performed in a high-throughput manner with a sample throughput of at least 60 samples h(-1). In addition, the adsorption of the analyte on the electrode surface was prevented due to compartmentalization in droplets. Linearity of the proposed system was found to be in the range of 0.5-5mM with a sensitivity of 7.42×10(-3)Amol(-1)Lcm(-2) and limits of detection and quantitation of 0.45 and 1.45mM, respectively. High intraday and interday (evaluated among 3 days) precisions for the detection of 50 droplets containing glucosamine were obtained with relative standard deviation less than 3%. The system was successfully used to determine the amounts of glucosamine in supplementary products with error percentage and relative standard deviation less than 3%. In addition, the amounts of glucosamine measured using the developed sensor were in good agreement with those obtained from a CE method. These indicate high accuracy and precision of the proposed system.

Graphene-polyaniline modified electrochemical droplet-based microfluidic sensor for high-throughput determination of 4-aminophenol.

We report herein the first development of graphene-polyaniline modified carbon paste electrode (G-PANI/CPE) coupled with droplet-based microfluidic sensor for high-throughput detection of 4-aminophenol (4-AP) in pharmaceutical paracetamol (PA) formulations. A simple T-junction microfluidic platform using an oil flow rate of 1.8 µL/min and an aqueous flow rate of 0.8 µL/min was used to produce aqueous testing microdroplets continuously. The microchannel was designed to extend the aqueous droplet to cover all 3 electrodes, allowing for electrochemical measurements in a single droplet. Parameters including flow rate, water fraction, and applied detection potential (Edet) were investigated to obtain optimal conditions. Using G-PANI/CPE significantly increased the current response for both cyclic voltammetric detections of ferri/ferrocyanide [Fe(CN)6](3-/4-) (10 times) and 4-AP (2 times), compared to an unmodified electrode. Using the optimized conditions in the droplet system, 4-AP in the presence of PA was selectively determined. The linear range of 4-AP was 50-500 µM (R(2) = 0.99), limit of detection (LOD, S/N = 3) was 15.68 µM, and limit of quantification (LOQ, S/N = 10) was 52.28 µM. Finally, the system was used to determine 4-AP spiked in commercial PA liquid samples and the amounts of 4-AP were found in good agreement with those obtained from the conventional capillary zone electrophoresis/UV-Visible spectrophotometry (CZE/UV-Vis). The proposed microfluidic device could be employed for a high-throughput screening (at least 60 samples h(-1)) of pharmaceutical purity requiring low sample and reagent consumption.

Determination of gibberellic acid in fermentation broth and commercial products by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) was developed as a method for quantitative determination of gibberellic acid (GA3) in fermentation broth and commercial products, using 25 mM disodium tetraborate as a buffer at pH 9.2 and 100 mM sodium dodecyl sulfate as a micellar phase. The baseline resolution (Rs of GA3 from other compounds in fermentation broth was achieved with Rs > 2.5. The addition of methanol or acetonitrile in the MEKC buffer did not give a better resolution. Advantages of this MEKC method include high accuracy and precision and no sample preparation except for dilution and filtration.

Electrochemical droplet-based microfluidics using chip-based carbon paste electrodes for high-throughput analysis in pharmaceutical applications.

This paper presents the first example of a pharmaceutical application of droplet-based microfluidics coupled with chronoamperometric detection using chip-based carbon paste electrodes (CPEs) for determination of dopamine (DA) and ascorbic acid (AA). Droplets were generated using an oil flow rate of 1.80 µL min(-1), whereas a flow rate of 0.80 µL min(-1) was applied to the aqueous phase, which resulted in a water fraction of 0.31. The optimum applied potential for chronoamperometric measurements in droplets was found to be 150 mV. Highly reproducible analysis of DA and AA was achieved with relative standard deviations of less than 5% for both intra-day and inter-day measurements. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 20 and 70 µM for DA and 41 and 137 µM for AA, respectively. Linearity of this method was in the ranges of 0.02-3.0mM for DA and 0.04-3.0mM for AA. This system was successfully applied to determine the amounts of DA and AA in intravenous drugs. Calibration curves of DA and AA for quantitative analysis were obtained with good linearity with R(2) values of 0.9984 and 0.9988, respectively. Compared with the labeled amounts, the measured concentrations of DA and AA obtained from this system were insignificantly different, with error percentages of less than ±3.0%, indicating a high accuracy of the developed method.

Calcium oxalate crystallization index (COCI): an alternative method for distinguishing nephrolithiasis patients from healthy individuals.

Urinary supersaturation triggers lithogenic crystal formation. We developed an alternative test, designated calcium oxalate crystallization index (COCI), to distinguish nephrolithiasis patients from healthy individuals based on their urinary crystallization capability. The effect of urine volume, oxalate, phosphate, citrate, potassium, and sodium on COCI values was investigated. COCI values were determined in 24-hr urine obtained from nephrolithiasis patients (n=72) and matched healthy controls (n=71). Increases in urine oxalate and phosphate and decreases in urine volume and citrate resulted in significantly increased COCI values. The urinary COCI in nephrolithiasis patients was significantly higher than that in healthy individuals. Two healthy subjects who had elevated COCI values were found to have asymptomatic kidney calculi. The receiver operating characteristic analysis showed an area under the curve of the urinary COCI test of 0.9499 (95%CI: 0.9131-0.9868) for distinguishing between nephrolithiasis and healthy subjects. At the cutoff of 165 mg oxalate equivalence/day, the urinary COCI test provided sensitivity, specificity, and accuracy amounts of 83.33%, 97.18%, and 90.21%, respectively. Urinary COCI values were primarily dependent on urine volume, oxalate, and phosphate. The test provided high sensitivity and specificity for clinically discriminating nephrolithiasis patients from healthy controls. It might be used to detect individuals with asymptomatic kidney calculi.