Innovative technology for evaluation of sperm DNA double-strand breaks diagnoses male factor infertility and prevents reproductive failures.

Neutral comet assay has been available for two decades to evaluate sperm double-strand breaks (DSBs). However, its clinical usability is limited due to its complex and time-consuming procedure, as well as the lack of a standardized scoring system. The aim of this study was to: develop a rapid diagnostic method for DSBs, Sperm DNA Fragmentation Releasing Assay (SDFR), and explore the association between DSBs and reproductive outcomes. We pioneered the use of polyacrylamide (PA) for embedding sperm chromatin and optimized the porosity of PA to be between 10 and 13%. The refined PA network allowed the trapping of DSBs, which dispersed halo on an immunological slide; in contrast, intact chromatin failed to develop a halo. A strong correlation was showed between reproducible values obtained from SDFR and neutral comet assay. SDFR were responsive to dose-/time-dependent simulated DSBs, indicating high sensitivity and specificity. Furthermore, we conducted a retrospective study of couples with embryonic aneuploidy screening, and recording DSB profiles of the male partners. Our findings revealed that DSB enabled to predict embryonic aneuploidy whereas basic semen parameters did not. In conclusion, SDFR offers a rapid and user-friendly approach for evaluating DSBs, with potential implications for predictive healthcare in reproductive medicine.

Live motile sperm sorting device for enhanced sperm-fertilization competency: comparative analysis with density-gradient centrifugation and microfluidic sperm sorting.

PURPOSE: A live motile sperm sorting device (LensHooke® CA0) developed to prevent the deleterious effects of centrifugation was evaluated comparatively with conventional density-gradient centrifugation (DGC) and microfluidic-based device (Zymot) in sperm selection. METHODS: Semen samples from 239 men were collected. CA0 under different incubation intervals (5, 10, 30, and 60 min) and temperatures (20, 25, and 37℃) was conducted. The sperm quality in CA0-, DGC-, and Zymot-processed samples was then comparatively evaluated. Semen parameters included concentration, motility, morphology, motion kinematics, DNA fragmentation index (DFI), and the rate of acrosome-reacted sperm (AR). RESULTS: Total motility and motile sperm concentration increased in a time- and temperature-dependent manner and the total motility peaked for 30 min at 37℃. In paired analysis, CA0 showed significantly higher total motility (94.0%), progressive motility (90.8%), rapid progressive motility (83.6%), normal morphology (10.3%), and lower DFI (2.4%) and AR (4.7%) than the other two methods in normozoospermic samples (all p < 0.05). For non-normozoospermic samples, CA0 had significantly better results than the other two methods (total motility 89.2%, progressive motility 80.4%, rapid progressive motility 74.2%, normal morphology 8.5%, DFI 4.0%, and AR 4.0%; all p < 0.05). CONCLUSION: CA0 yielded spermatozoa with enhanced sperm fertilization potentials; DFI was minimized in samples processed by CA0. CA0 was effective for both normal and abnormal semen samples due to its consistent selection efficiency.

Development and integration of LensHooke® R10 for automatic and standardized diagnosis for sperm DNA fragmentation.

BACKGROUND: The sperm chromatin dispersion assay is commonly used to assess sperm DNA integrity. This approach is time-consuming, demonstrates poor chromatin preservation, and provides an ambiguous and unstandardized evaluation of fragmented chromatin. OBJECTIVES: We aimed to (i) develop an optimized sperm chromatin dispersion assay with reduced operation time, (ii) validate R10 test accuracy by comparing it to a conventional sperm chromatin dispersion assay, and (iii) standardize the sperm DNA fragmentation analysis procedure by integrating artificial intelligence optical microscopic technology. MATERIALS AND METHODS: This cross-section study included 620 semen samples. Aliquots were analyzed by a conventional Halosperm® G2 assay (G2) and LensHooke® R10 assay (R10). The DNA fragmentation index was scored manually, and R10 slides were automatically determined by a LensHooke® X12 PRO semen analysis system (X12). RESULTS: We demonstrated significant improvements in total assay time (40 vs. 72 min, p < 0.001) and in the halo-cytological resolution using R10 compared to G2. Comparing the G2 and R10, DNA fragmentation index results demonstrated good agreement between the two methods (Spearman's rank correlation, rho = 0.8517, p < 0.0001). We introduced the integration of an auto-calculation system to diagnose sperm DNA fragmentation. X12 interpretation showed excellent agreement with manual interpretation (Spearman's rank correlation, rho = 0.9323, p < 0.0001), but had a low coefficient of variation compared to manual interpretation (4% for R10 by X12 vs. 19% for R10 by manual scoring vs. 25% for G2 by manual scoring). DNA fragmentation index was more correlated with total motility (coefficients = -0.3607, p < 0.0001) than sperm morphology and was positively associated with asthenozoospermic semen samples (p = 0.0001). CONCLUSION: The R10 sperm chromatin dispersion assay combined with the X12 semen analysis system is faster, more objective, and provides standardization for sperm DNA fragmentation.

Testing quality of a self-monitoring blood glucose sensor with an auto-coding mechanism when used by patients versus technicians.

BACKGROUND: In response to the problem of erroneous readings due to miscoding when performing self-monitoring blood glucose (SMBG), this study introduces a user-friendly SMBG biosensor with an innovative auto-coding module on the meter and strip. Actual users characterized the performance of the SMBG systems. METHODS: A total of 105 patients were incorporated in the study and Clarke error grid analysis (EGA) was administered to evaluate the clinical accuracy of the results obtained by the patients versus the technicians. All patients used the questionnaires to comment on the use of the auto-coding sensor. RESULTS: In the imprecision test, the total CV of the 5 BG levels was 2.1%. In the EGA plot, the results of the auto-coding sensor were 96.2%, both lots A and B, in zone A for the patients and 99.0% and 97.1% for the technician. The paired t-test demonstrated no statistically significant difference between the patient and technician measurements. Regression analysis also demonstrated that the measurements taken by the patients agreed with those obtained using the laboratory method. CONCLUSIONS: The patients achieved satisfactory performance using the auto-coding SMBG sensor and derived similar results with both laboratory reference and operation by a technician.

Evaluation of accuracy of FAD-GDH- and mutant Q-GDH-based blood glucose monitors in multi-patient populations.

BACKGROUND: Glucose dehydrogenases have been highly promoted to high-accuracy blood glucose (BG) monitors. The flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH) and mutant variant of quinoprotein glucose dehydrogenase (Mut. Q-GDH) are widely used in high-performance BG monitors for multi-patient use. Therefore we conducted accuracy evaluation of the GDH monitors, FAD-GDH-based GM700 and Mut. Q-GDH-based Performa. METHODS: Different patients were enrolled: patients with and without diabetes, patients receiving respiratory therapies, hemodialysis (HD) and peritoneal dialysis (PD) patients, and neonates. The accuracy evaluation of FAD-GDH- and Mut. Q-GDH-based monitors referred to ISO 15197:2013 which applies new criteria for the minion accuracy requirements: more than 95% of the blood glucose readings shall fall within ±15mg/dL of the reference method at glucose concentration <100mg/dL and within ±15% of the reference method at glucose concentration ≥100mg/dL. Bland-Altman plots were used to evaluate the 2 GDH monitors as well. RESULTS: Bland-Altman plots visualized excellent precision of the BG monitors. The 95% limit agreement of overall results for the FAD-GDH-based monitors was within ±12% and that for the Mut. Q-GDH-based monitors was from -10 to +17%. Both BG monitors met the accuracy requirements of ISO 15197:2013. The FAD-GDH-based monitor performed better with neonates and patients with and without diabetes, and the Mut. Q-GDH-based monitor performed better with HD and PD patients. CONCLUSIONS: Analytical results prove that the GDH-based monitors tolerate a broad BG concentration range, are oxygen independent, have BG specificity, and have minimal interference from hematocrit. The GDH-based monitors are reliable for multi-patient use.

Accuracy and precision evaluation of seven self-monitoring blood glucose systems.

BACKGROUND: Self-monitoring blood glucose (SMBG) systems play a critical role in management of diabetes. SMBG systems should at least meet the minimal requirement of the World Health Organization's ISO 15197:2003. For tight glycemic control, a tighter accuracy requirement is needed. METHODS: Seven SMBG systems were evaluated for accuracy and precision: Bionime Rightest(™) GM550 (Bionime Corp., Dali City, Taiwan), Accu-Chek(®) Performa (Roche Diagnostics, Indianapolis, IN), OneTouch(®) Ultra(®)2 (LifeScan Inc., Milpitas, CA), MediSense(®) Optium(™) Xceed (Abbott Diabetes Care Inc., Alameda, CA), Medisafe (TERUMO Corp., Tokyo, Japan), Fora(®) TD4227 (Taidac Technology Corp., Wugu Township, Taiwan), and Ascensia Contour(®) (Bayer HealthCare LLC, Mishawaka, IN). The 107 participants (44 men and 63 women) were between 23 and 91 years old. The analytical results of seven SMBG systems were compared with those of plasma analyzed with the hexokinase method (Olympus AU640, Olympus America Inc., Center Valley, PA). RESULTS: The imprecision of the seven blood glucose meters ranged from 1.1% to 4.7%. Three of the seven blood glucose meters (42.9%) fulfilled the minimum accuracy criteria of ISO 15197:2003. The mean absolute relative error value for each blood glucose meter was calculated and ranged from 6.5% to 12.0%. CONCLUSIONS: More than 40% of evaluated SMBG systems meet the minimal accuracy criteria requirement of ISO 15197:2003. However, considering tighter criteria for accuracy of ±15%, only the Bionime Rightest GM550 meets this requirement. Because SMBG systems play a critical role in management of diabetes, manufacturers have to strive to improve accuracy and precision and to ensure the good quality of blood glucose meters and test strips.

Superior long-term stability of a glucose biosensor based on inserted barrel plating gold electrodes.

Disposable one shot usage blood glucose strips are routinely used in the diagnosis and management of diabetes mellitus and their performance can vary greatly. In this paper we critically evaluated the long-term stability of glucose strips made of barrel plating gold electrodes. Compared to other glucose biosensing platforms of vapor deposited palladium and screen printed carbon electrodes, the proposed glucose biosensor was found to show the best stability among the three biosensing platforms in thermal acceleration experiments at 40 degrees C for 6 months with an average bias of 3.4% at glucose concentrations of 5-20 mM. The precision test of this barrel plating gold glucose biosensor also showed the best performance (coefficients of variation in the range of 1.4-2.4%) in thermal acceleration experiments at 40 degrees C, 50 degrees C and 70 degrees C for 27 days. Error grid analysis revealed that all measurements fell in zone A and zone B. Regression analysis showed no significant difference between the proposed biosensor and the reference method at 99% confidence level. The amperometric glucose biosensor fabricated by inserting two barrel plating gold electrodes onto an injection-molding plastic base followed by immobilizing with a bio-reagent layer and membrane was very impressive with a long-term stability up to 2.5 years at 25 degrees C. Overall, these results indicated that the glucose oxidase/barrel plating gold biosensing platform is ideal for long-term accurate glycemic control.

Multiple screening of urolithic organic acids with copper nanoparticle-plated electrode: potential assessment of urolithic risks.

There is yet to be a reliable prediction of urolithiasis. To facilitate early diagnosis, a simple and rapid high performance liquid chromatography method with electrochemical detection using disposable copper-nanoparticle-plated electrodes (Cu(n)-SPE) was developed for multiple detection of creatinine and 4 urolithic organic acids. A total of 206 normal and urolithic human and canine urines and urolith samples were collected for direct analysis of creatinine, cystine, uric acid, oxalic acid, and citric acid without sample cleanup and derivatization processes. Urinary organic acids were separated in 11 min and were devoid of ascorbic acid interference. The detection limits (S/N>3) were at the nanomolar level with linear dynamic ranges spanning 2-3 orders of magnitude. Recoveries in urine ranged from 99.5% for creatinine to 86.5% for citric acid. The analytical variations (RSD) were less than 6.2% in phosphate buffer and 7.7% in urine. Important differences in organic acid levels/profiles between animal species and among normal and urolithic urines/urolith were unveiled and corresponded well (70-90%) with the urolithic risk in a retrospective assessment. The simplicity and reproducibility of this method using disposable Cu(n)-SPE has made routine urine analysis possible and can be of great clinical and diagnostic potential in the screening of urolithiasis and abnormal states related to excess secretion of organic acids and amino acids in humans and animals.

A disposable screen-printed silver strip sensor for single drop analysis of halide in biological samples.

A screen-printed silver strip with three-electrode configuration of Ag-working, Ag-counter and Ag/Ag(x)O reference electrodes was developed for simultaneous determination of chloride, bromide and iodide in aqueous solutions. It was fabricated simply by screen-printing silver ink onto a polypropylene (PP) base. The in-situ prepared Ag/Ag(x)O reference electrode can avoid the leaching interference in chloride detection while using a conventional Ag/AgCl reference electrode. A single drop of analyte (50 microl) is enough to determine iodide, bromide and chloride by measuring the well-separated oxidation peak currents of respective silver halides. The calibration graph was linear from 10 microM to 20 mM for iodide and bromide and 100 microM to 20 mM for chloride and the detection limit (S/N=3) was 3.05 microM, 2.95 microM and 18.83 microM for iodide, bromide and chloride, respectively. The strip is designed to be disposable and as such manual polishing is not necessary. The proposed sensor is not only simple to manufacture and easy to operate but also fast and precise with little detection volume. It is successfully applied to the determination of halide ions in real samples.

Assessing the quality of Bionime self-monitoring blood glucose system Rightest GM110: a critical evaluation of interference and ambient circumstances.

BACKGROUND: The key issue in preventing chronic diabetic complications is to maintain near-normoglycemia. Analytical evaluation of Bionime self-monitoring blood glucose (SMBG) Rightest GM110 was carried out in this study. METHODS: The evaluation was executed according to the Standards for Reporting Diagnostic Accuracy (STARD) and the Clinical and Laboratory Standards Institute (CLSI). The evaluation procedure mainly focused on analytical performance. The accuracy tests included hematocrit, interferants, temperature, humidity, altitude and clinical evaluations. RESULTS: Good linearity response (R(2)>0.99) and satisfactory precision (CVs: 1.1-2.8%) were observed in glucose concentrations of 0.6-30.5 mmol/l. In hematocrit test, the Rightest GM110 was suitable for use in sample containing hematocrit in the range of 30-55%. Interfering test indicated that almost all substances tested were insignificant, with bias <10% in medium- and hyper-glycemia samples. Satisfactory stability was also found under various ambient circumstances, with bias within +/-10%. In clinical trials, values within the acceptable zone (A+B) were 100% and values within zone A exceed 95% in error grid analysis. CONCLUSIONS: The Bionime Rightest GM110 is reliable to display accurate glucose concentrations in specimens with irresistible interfering factors.

Fabrication of a glucose biosensor based on inserted barrel plating gold electrodes.

We demonstrate here the application of barrel plating gold electrodes for fabricating a new type of disposable amperometric glucose biosensor. It is prepared by inserting two barrel plating gold electrodes onto an injection molding plastic base followed by immobilizing with a bioreagent layer and membrane on the electrode surface. The primary function of barrel plating is to provide an economical way to electroplate manufactured parts. The manufacture procedure is simple and can increase the fabrication precision for automation in mass production. At the two-electrode system, the detection of glucose is linear up to 800 mg/dL (i.e., 44.5 mM, r(2) > 0.99) in pH 7.4 PBS with a sensitivity of 0.71 microA/mM. Excellent sensor-to-sensor reproducibility shows coefficients of variation of only 0.8-1.4% for the detection of 56.5-561.0 mg/dL glucose. In laboratory trials 176 capillary blood samples with a range of 30-572 mg/dL glucose are used to evaluate the clinical application of the biosensor. A good linear correlation is observed between the measured values of the proposed biosensor and laboratory reference. Error grid analysis verifies that the proposed technique is promising in fabricating biosensor strips on a mass scale. As successfully demonstrated by using whole blood glucose as a model analyte, the fabrication technique can extend into other barrel plating noble metal electrodes for various applications.

Clinical evaluation of bionime rightest GM310 biosensors with a simplified electrode fabrication for alternative-site blood glucose tests.

BACKGROUND: Most processes for fabricating biosensors applied to screen-printed carbon electrodes (SPCEs) are complex. This study presents a novel one-step process for manufacturing electrodes for injection-molding biosensors. METHODS: During the sensor-fabrication process, barrel-plated gold electrodes were inserted into an injection-molded base. The electrode directly touched the electrical contact of a meter. We analyzed technical measurements for this biosensor, including tests of the measurement range, within-run imprecision, and between-meter imprecision. In clinical trials, experienced technicians tested 3 alternative sites (fingertip, palm, and arm). The results were simultaneously compared with plasma values obtained with the hexokinase method on the Olympus AU640 instrument. Analytical results were evaluated according to International Standards Organization 15197 (ISO 15197:2003) criteria and by Clarke error grid analysis (EGA), and CVs were calculated to evaluate within-run imprecision. RESULTS: The glucose measurement range was 0.6- 33.3 mmol/L (y = 0.96x + 0.07 mmol/L; r(2) = 0.9977). The CVs in the within-run imprecision test were 1.7%-3.5%, and the overall CV was 2.1%, indicating good reproducibility of results. The Student t-tests of mean values from 5 meters revealed statistically insignificant differences (P > 0.05). In clinical trials, the agreement of the Rightest GM310 meter results with those of a laboratory method complied with ISO 15197:2003 criteria. In the EGA, 100% of the values were within the acceptable zones (A + B), and the proportion of values within zone A exceeded 95%. CONCLUSIONS: The Bionime Rightest GM310 meter applied a simplified process for biosensor fabrication and displayed acceptable performance for monitoring glucose concentrations at alternative test sites.

Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes.

A simple, rapid and reliable method based on high-performance liquid chromatography with electrochemical detection was developed to routinely differentiate among meat products from fifteen food animal species. Samples from cattle, pigs, goats, deer, horses, chickens, ducks, ostriches, salmon, cod, shrimp, crabs, scallops, bullfrogs and alligators each exhibited unique electrochemical profiles. Species-specific markers exhibited reproducible peak retention times with coefficients of variation less then 6% across different runs, body regions and subjects. The method requires no derivatization or extraction steps and may be applicable to fresh or cooked meats. Incubation of fresh beef, pork or chicken at room temperature for 24h or repeated freezing and thawing changed the intensity but not the pattern of species-specific peaks. In conclusion, this method appears suitable for rapid differentiation of meats from various food animal species and demonstrates the utility of electrochemical detection to supplement existing immunochemical and molecular biological methods. The possibility of using this method to detect adulteration and degradative changes of meat proteins is discussed.

An electrochemical cell coupled with disposable screen-printed electrodes for use in flow injection analysis.

An electrochemical cell coupled with disposable screen-printed electrodes (SPEs) that is specifically designed for use in flow injection analysis (FIA) is described in this study. The cell is made of foldable polyoxymethylene (acetal) thick platelets with the bottom portion consisting of a cavity track to drag the SPEs in position and the top portion having predrilled T-like holes to arrange the Ag/AgCl reference electrode and stainless steel inlet & outlet. An "O ring" is suitably fixed on the top of the working electrode to form a thin-layer space where the electrochemical reaction can take place. Hydrodynamic characterization was validated by using a benchmark hexacyanoferrate redox couple. The results of practical analysis of glucose in human plasma clearly demonstrate the characteristics and applicability of the proposed wall-jet electrochemical cell in FIA.

Amino acid analysis using disposable copper nanoparticle plated electrodes.

A disposable copper nanoparticle-plated screen-printed carbon electrode (designated as Cun-SPE(100-nm)) provides a new material for the determination of native amino acids. All 20 underivatized amino acids can be sensitively determined at 0.0 V vs. Ag/AgCl in pH 8 phosphate buffer solution. The precisely controlled copper nanoparticles can boost up the CuIIO/CuI2O redox signal on the working surface without any prior pretreatment procedure. The formation of a reversible 1:1 CuIIO-amino acid complex on the Cun-SPE(100-nm) was proposed to play a key role in the reaction mechanism. Stable detection responses were obtained for all amino acids by flow injection analysis with detection limits (S/N = 3) that lie in the range of 24 nM-2.7 microM. Selected amino acids from six representative chemical natures were separated by HPLC and detected at the Cun-SPE(100-nm) with promising results.

Voltammetric peak separation of dopamine from uric acid in the presence of ascorbic acid at greater than ambient solution temperatures.

Peak overlap in voltammetry poses challenges for the quantitative analysis of electroactive species. Dopamine and uric acid are typically challenging to determine voltammetrically because of their very similar oxidation peak potentials. We report preliminary results of the use of a screen-printed carbon electrode for the determination of dopamine and uric acid in an electrolyte solution maintained above ambient temperatures. Higher temperatures resulted in dramatic shifting of the dopamine oxidation peak toward lower potentials, while the uric acid peak was essentially stationary. Ascorbic acid, an interference in voltammetric uric acid determinations, is effectively suppressed at higher temperatures. This resulted in a greater peak separation of dopamine from uric acid at higher temperatures, which is desirable for better peak integration. In addition, greater current responses for both species were recorded at higher temperatures. The cause for such an increase in peak current is unraveled using ac impedance measurements. Presented are preliminary results for determining dopamine and uric acid at temperatures higher than ambient. Much improved voltammetric peak separation and sensitivity is obtained at these higher temperatures compared to ambient.

Photoelectrochemical oxygen sensor using copper-plated screen-printed carbon electrodes.

We report here an efficient photocatalytic amperometric sensor for the determination of dissolved oxygen (DO) in phosphate buffer solution using a disposable copper-plated screen-printed carbon electrode (CuSPE). The photoelectrochemical activity toward DO of the CuSPE was related to the formation of a p-type semiconductor Cu(I)2O. The solution pH and biased potential (E(bias)) were systematically optimized as pH 8 PBS and -0.7 V vs Ag/AgCl, respectively. Under optimized conditions, the calibration plot was linear in the range of 1-8 ppm with sensitivity and regression coefficient of 23.51 (microA cm2)(-1) ppm(-1) and 0.9982, respectively. The reproducibility of the system was good with seven successive measurements of DO yielding a RSD value of 1.87%. Real sample assays for groundwater and tap water were also consistent with those measured by a commercial DO meter. The principle used in DO measurement has an opportunity to extend into various research fields.