Magnetoelastic Immunosensor via Antibody Immobilization for the Specific Detection of Lysozymes.

Lysozymes in human urine have crucial clinical significance as an indicator of renal tubular and glomerular diseases. Most lysozyme detection methods rely on the enzyme-linked immunosorbent assay (ELISA), which is usually a tedious procedure. Meanwhile, aptamer sensors and fluorescence-based techniques for lysozyme detection have emerged in recent studies. However, these methods are time-consuming and highly complex in operation, and some even require exorbitant reagents and instruments, which restricts real-time clinical monitoring as diagnostic approaches. Therefore, a rapid and low-cost lysozyme detection method with facile preparation is still in demand for modern precision medicine. Herein, we propose a magnetoelastic (ME) immunosensor for lysozyme detection by detecting changes in resonance frequency under a magnetostrictive effect. The detection system is composed of a magnetoelastic chip with an immobilized lysozyme antibody, a solenoid coil, and a vector network analyzer. Since the ME sensor is ultrasensitive to mass change, the frequency offset caused by mass change can be utilized to detect the content of lysozyme. The immunosensor is evaluated to possess superior sensitivity of 138 Hz/µg mL-1 in terms of the resonance frequency shift (RFS). In addition, our sensor displays an outstanding performance in specificity experiments and shows a relatively lower detection limit (1.26 ng/mL) than other conventional lysozyme detection methods (such as ELISA, chemiluminescence assay, fluorescence, and aptamer biosensors).

Cystatin C, Neutrophil Gelatinase-associated Lipocalin, and Lysozyme C: Urinary Biomarkers for Detection of Early Kidney Dysfunction in Children With Urolithiasis.

OBJECTIVE: To screen for the presence of biomarkers involved in tubular injury and kidney damage in children with urolithiasis (RS), and to validate these proteins by ELISA. METHODS: Prospective-controlled pilot study of children with urolithiasis and their age- and gender-matched controls (HC). Initial screening test was done by quantitative proteomic comparison of pooled urine from RS versus HC, using liquid chromatography-mass spectrometry. Proteins of interest were selected using the following criteria: (1) ≥5 spectral counts; (2) ≥2-fold difference in spectral counts; and (3) ≤.05 P value for the Fisher's Exact Test. Validation was performed by ELISA testing. Statistical analysis was performed by Student t-test and Mann-Whitney U test. RESULTS: Proteomic analysis identified 3 proteins of interest, Cystatin C (CYTC), neutrophil gelatinase-associated lipocalin (NGAL) and lysozyme C that were significantly over-represented in RS group versus HC. ELISA analysis revealed significantly increased urinary levels of CYTC and NGAL, and nearly significantly increased urinary levels of lysozyme C in RS group (N = 24) compared to controls (N = 13). Subgroup analysis showed significantly higher urinary levels of CYTC in both hypercalciuria (N = 14) and hypocitraturia (N = 10) versus HC (P <.05). CONCLUSION: Children with urolithiasis showed significant increase in urinary CYTC and NGAL irrespective of their normal serum creatinine. These biomarkers indicate tubular injury and early kidney damage and represent valid tools for early screening when traditional tests are normal.

Fabrication and evaluation of molecularly imprinted magnetic nanoparticles for selective recognition and magnetic separation of lysozyme in human urine.

In this study, lysozyme-based magnetic molecularly imprinted polymers (Lyz-MMIPs) for selective recognition and magnetic separation of lysozyme in human urine were prepared via surface imprinting technology. The morphology and structural properties of the resultant Lyz-MMIPs were characterized by using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), thermogravimetry (TG), X-ray diffraction (XRD) and a vibrating sample magnetometer (VSM). The results showed that the Lyz-MMIPs exhibited a uniform core-shell structure and favorable magnetic properties with a saturation magnetization of 14.8 emu g-1. To obtain the best selectivity and binding performance, the pH value of adsorption solution was investigated in detail. Under the optimized conditions, the Lyz-MMIPs possessed high binding and specific recognition capacity, as well as fast binding kinetics and phase separation rates. Moreover, the saturation binding capacity of Lyz-MMIPs reached 124.3 mg g-1, which was nearly 3.2 times that of lysozyme-based magnetic non-imprinted polymers (Lyz-MNIPs). In addition, the selectivity and reusability experiments showed that the Lyz-MMIPs displayed significant selectivity and favorable reusability. Furthermore, the Lyz-MMIPs were successfully applied for the determination and separation of lysozyme in human urine with satisfactory recovery rates. Above all, the synthetic process was quite simple and this strategy may provide a versatile approach for the fabrication of well-defined molecularly imprinted polymers on magnetic nanoparticles for the analysis of complicated matrixes.

Proteomic Analysis of Urine from California Sea Lions ( Zalophus californianus): A Resource for Urinary Biomarker Discovery.

Urinary markers for the assessment of kidney diseases in wild animals are limited, in part, due to the lack of urinary proteome data, especially for marine mammals. One of the most prevalent kidney diseases in marine mammals is caused by Leptospira interrogans, which is the second most common etiology linked to stranding of California sea lions ( Zalophus californianus). Urine proteins from 11 sea lions with leptospirosis kidney disease and eight sea lions without leptospirosis or kidney disease were analyzed using shotgun proteomics. In total, 2694 protein groups were identified, and 316 were differentially abundant between groups. Major urine proteins in sea lions were similar to major urine proteins in dogs and humans except for the preponderance of resistin, lysozyme C, and PDZ domain containing 1, which appear to be over-represented. Previously reported urine protein markers of kidney injury in humans and animals were also identified. Notably, neutrophil gelatinase-associated lipocalin, osteopontin, and epidermal fatty acid binding protein were elevated over 20-fold in the leptospirosis-infected sea lions. Consistent with leptospirosis infection in rodents, urinary proteins associated with the renin-angiotensin system were depressed, including neprilysin. This study represents a foundation from which to explore the clinical use of urinary protein markers in California sea lions.

Lysozyme aptasensor based on a glassy carbon electrode modified with a nanocomposite consisting of multi-walled carbon nanotubes, poly(diallyl dimethyl ammonium chloride) and carbon quantum dots.

An aptamer-based method is described for electrochemical determination of lysozyme. A glassy carbon electrode was modified with a nanocomposite composed of multi-walled carbon nanotubes, poly(diallyl dimethyl ammonium chloride), and carbon quantum dots. The composition of the nanocomposite (MWCNT/PDDA/CQD) warrants good electrical conductivity and a high surface-to-volume ratio. The lysozyme-binding aptamers were immobilized on the nanocomposite via covalent coupling between the amino groups of the aptamer and the carboxy groups of the nanocomposite. The modified electrode was characterized by electrochemical impedance spectroscopy, cyclic voltammetry and differential pulse voltammetry. The use of this nanocomposite results in a considerable enhancement of the electrochemical signal and contributes to improving sensitivity. Hexacyanoferrate was used as an electrochemical probe to study the dependence of the peak current on lysozyme concentration. In the presence of lysozyme, the interaction of lysozyme with immobilized aptamer results in a decrease of the peak current, best measured at +0.15 V vs. Ag/AgCl. A plot of peak current changes versus the logarithm of the lysozyme concentration is linear in the 50 fmol L-1 to 10 nmol L-1 concentration range, with a 12.9 fmol L-1 detection limit (at an S/N ratio of 3). The method is highly reproducible, specific and sensitive, and the electrode has a rapid response. It was applied to the determination of lysozyme in egg white, serum, and urine. Graphical abstract Schematic of a nanocomposite composed of multi-walled carbon nanotubes (MWCNTs), poly(diallyldimethyl ammonium chloride) (PDDA), and carbon quantum dots (CQDs) for use in a lysozyme aptasensor. The aptamer was immobilized on the surface, and bovine serum albumin (BSA) was applied to block the surface. The changes of peak current for the electrochemical probe hexacyanoferrate (Fe(CN)63-/4-) in the presence and absence of lysozyme was traced.

Novel oligo(ethylene glycol)-based molecularly imprinted magnetic nanoparticles for thermally modulated capture and release of lysozyme.

In this study, oligo(ethylene glycol) (OEG)-based thermoresponsive molecularly imprinted polymers (MIPs) for lysozyme on the surface of magnetic nanoparticles were synthesized. Thermoresponsive monomer 2-(2-methoxyethoxy)ethyl methacrylate, chelate monomer N-(4-vinyl)-benzyl iminodiacetic acid, and acidic monomer methacrylic acid were selected as the ingredients for preparing the MIP layer. The thermoresponsive behavior of the novel imprinted magnetic nanoparticles was evaluated by dynamic light scattering and swelling ratios measurements. Interestingly, in analysis of lysozyme, the capture/release process could be modulated by changing the temperature, avoiding tedious washing steps. Meanwhile, high adsorption capacity (204.1 mg/g) and good selectivity for capturing lysozyme were achieved. Additionally, surface imprinting with magnetic nanoparticles as substrate allowed for short adsorption time (2 h) and rapid magnetic separation. Furthermore, the proposed imprinted magnetic nanoparticles were used to selectively extract lysozyme in human urine with recoveries ranging from 89.2% to 97.3%. The results indicated that the OEG-based monomers are promising for responsive MIP preparation, and the proposed imprinted material is efficient for thermally modulated capture and release of target protein.

Non-covalent conjugation of CdTe QDs with lysozyme binding DNA for fluorescent sensing of lysozyme in complex biological sample.

Water-soluble cysteamine (CA) capped CdTe quantum dots (QDs) conjugated with lysozyme binding DNA (LBD) was constructed for luminescent sensing of lysozyme by forming a ternary self-assembly complex. Addition of negatively charged lysozyme binding DNA to the positively charged CA capped CdTe QDs buffer solution (Tris-HCl pH 7.4) could lead to the formation of QDs-LBD complex through electrostatic interactions. Once lysozyme was introduced into the CdTe QDs-LBD system, it could bind specifically with the QDs-LBD complex, resulting in fluorescence emission enhancement of the QDs due to the surface inert of QDs. At a given amount of LBD and CdTe QDs (LBD: QDs=2: 1), the fluorescence intensity enhancement of QDs was linear with lysozyme concentration over the range of 8.9-71.2 nM, with a detection limit of 4.3 nM. Due to the specific binding of LBD with lysozyme, this approach displayed high selectivity for lysozyme recognition. The sensing mechanism was confirmed by DLS and zeta potential measurement, and agarose gel electrophoresis experiment. Furthermore, the proposed CA-capped CdTe QDs-LBD sensor was applied to lysozyme detection in mouse serum and human morning urine samples, which showed high sensitivity and selectivity in the complex biological sample.

Hydrophobically modified quaternized celluloses as new dynamic coatings in CE for basic protein separation.

Hydrophobically modified quaternized celluloses (HMQCs), containing the quaternary ammonium groups and hexadecyl groups, were homogeneously synthesized as novel dynamic coatings for CE. Compared with quaternized cellulose coating, HMQC coating is able to generate stronger reversed EOF (ca. 10% increase) and has better efficiency in suppressing protein adsorption. The effects of the polymer concentration, the degree of hydrophobic substitution, and the buffer pH on the EOF mobility as well as on the separation of basic proteins were investigated in detail. It was shown that the use of HMQC coating could obviously improve the separation performance of basic proteins within a broad pH range. Five basic proteins (lysozyme, ribonuclease A, cytochrome c, bovine pancreatic trypsin inhibitor, and chymotrypsinogen) could be completely separated at pH 8.0. The successful performance of HMQC was further demonstrated by the analyses of lysozyme in tear and urine samples.

Rapid and selective determination of urinary lysozyme based on magnetic molecularly imprinted polymers extraction followed by chemiluminescence detection.

A rapid, low cost and selective chemiluminescence method coupled with magnetic molecularly imprinted polymers extraction was developed to detect lysozyme in human urine samples. Compared with traditional solid-phase extraction, this method could achieve selective extraction for the lysozyme, avoid the time consuming elution from a column or centrifugation steps, and then showed great potential in the high-throughput screening of clinical samples. The parameters affecting the performance of extraction and chemiluminescence were investigated. Under optimal conditions, the whole analytical procedure was completed within 12 min and spiked recovery ranged from 90.1% to 103.7% (R.S.D.≤6.7%). The limit of quantitation was 5 ng mL(-1). Furthermore, the results obtained by the proposed method were linearly correlated to those by commercial lysozyme detection kit (r=0.9595). Finally, the validated method was used to measure the urinary lysozyme of renal disease patients and healthy controls. The results confirmed the reliability and practicality of the protocol and revealed a good perspective of this method for biological sample analysis.

New strategy for label-free and time-resolved luminescent assay of protein: conjugate Eu3+ complex and aptamer-wrapped carbon nanotubes.

We report here a carbon nanotube-based approach for label-free and time-resolved luminescent assay of lysozyme (LYS) by engineering an antilysozyme aptamer and luminescent europium(III) (Eu(3+)) complex. The sensing mechanism of the approach is based on the exceptional quenching capability of carbon nanotubes for the proximate luminescent Eu(3+) complex and different propensities of single-stranded DNA and the DNA/protein complex to adsorb on carbon nanotubes. The luminescence of a mixture of chlorosulfonylated tetradentate ß-diketone-Eu(3+) and the antilysozyme aptamer was efficiently quenched by single-walled carbon nanotubes (SWNTs) unless the aptamer interacted with LYS. Due to the highly specific recognition ability of the aptamer for the target and the powerful quenching property of SWNTs for luminescence regents, this proposed approach has a good selectivity and high sensitivity for LYS. In the optimum conditions described, >700-fold signal enhancement was achieved for micromolar LYS, and a limit of detection as low as 0.9 nM was obtained, which is about 60-fold lower than those of commonly used fluorescent aptamer sensors. Moreover, due to the much longer lifetime of the Eu(3+) luminescence than those of the ubiquitous endogenous fluorescent components, the time-resolved luminescence technique could be conveniently used for application in complicated biological samples. LYS concentrations in human urine were thus detected using time-resolved luminescence measurement with satisfactory recoveries of 95-98%.

Direct determination of urinary lysozyme using surface plasmon resonance light-scattering of gold nanoparticles.

The purpose of this study was to establish a simple and sensitive analytical method for lysozyme using Plasmon Resonance Light-Scattering (PRLS) technique with Gold Nanoparticles (AuNPs) as the probe. Nanomolar level of lysozyme induced AuNPs aggregation with enhanced PRLS. For 1.4 nM citrate-capped AuNPs (13 nm in diameter), the linear range of the calibration curve was 15-50 nM with a detection limit of 13.1 nM for lysozyme. Six nanomolar lysozyme can produce an observable PRLS enhancement. Most potential interfering substances present in urine had a negligible effect on the determination. The interference from human serum albumin in the urinary sample can be reduced by precipitating the albumin with ethanol at pH 4.8-4.9. The 90.1-118.2% recovery was achieved for 8 individual lysozyme-spiked urinary samples. This simple and sensitive method for lysozyme does not require sample clean-up and AuNPs modification, thus provided an alternative for urinary lysozyme determination.

Synthesis of magnetic molecularly imprinted poly(ethylene-co-vinyl alcohol) nanoparticles and their uses in the extraction and sensing of target molecules in urine.

Superparamagnetic nanoparticles are of great current interest for biomedical applications in both diagnostics and treatment. Magnetic nanoparticles (MNP) can be manipulated by magnetic fields, so that when functionalized, they can be used for the purification and separation of biomolecules and even whole cells. Here we report combining the separation capabilities of MNPs with the functional (binding) capability of molecularly imprinted polymers. Albumin- creatinine-, lysozyme-, and urea-imprinted polymer nanoparticles were synthesized from poly(ethylene-co-ethylene alcohol) via phase inversion, with both target molecules and hydrophobic magnetic nanoparticles mixed within the polymer solution. Several ethylene:ethylene alcohol mole ratios were studied. The rebinding capacities for those three target molecules varied from 0.76 +/- 0.02 to 5.97 +/- 0.04 mg/g of molecularly imprinted magnetic nanoparticles. Lastly, the composite nanoparticles were used for separation and sensing of template molecules (e.g., human serum albumin) in real samples (urine) and results were compared with a commercial ARCHITECT ci 8200 system.

High serum and urine lysozyme levels in a dog with acute myeloid leukemia.

A 2-year-old, female German Shepherd Dog with facial nerve paralysis was diagnosed with acute myelomonocytic leukemia based on clinical, cytologic, and immunologic findings. Proteinuria (urine proteincreatinine ratio = 1.5) occurred in the absence of renal failure. Qualitative assessment of proteinuria by sodium dodecyl sulfate-agarose gel electrophoresis revealed a broad band with a molecular weight of approximately 15 kDa that was compatible with lysozyme (LZM). A diagnosis of tubular proteinuria was made, and a chemical evaluation of LZM in serum and urine samples was performed using a turbidimetric assay. The LZM concentrations were 24.5 mg/l (reference interval: 2.5-8.0 mg/l) and 274.5 mg/l (reference interval: <2 mg/l) in serum and urine, respectively.

Instant formation of molecularly imprinted poly(ethylene-co-vinyl alcohol)/quantum dot composite nanoparticles and their use in one-pot urinalysis.

The high stability of quantum dots (QDots) with photoluminescence has led to their increased use as imaging approaches in biological systems to replace conventional fluorescence labels. The antibodies are generally coated on the surface of QDots to the targeting site, and molecular imprinting polymers are designed to mimic the antibodies. Hence, quantum dots can be incorporated into molecularly imprinted polymers, which provide shape and selectivity, and then respond to template rebinding by emitting quenched photoluminescence. In this study, poly(ethylene-co-ethylene alcohol) creatinine-, albumin- and lysozyme-imprinted polymers nanoparticles are synthesized via phase inversion of poly(ethylene-co-ethylene alcohol) with various ethylene mole ratios when target molecules and hydrophobic quantum dots are mixed within the polymer solution. Finally, those particles were prepared for the detection of creatinine, human serum albumin and lysozyme in real sample (urine) and compared with commercial ARCHITECT ci 8200 system.

Acute health effects on planters of conifer seedlings treated with insecticides.

OBJECTIVES: The aim of this study was to assess acute health effects on planters caused by planting conifer seedlings treated with two insecticides, with active ingredients imidacloprid and cypermethrin, in comparison with untreated seedlings. METHODS: The investigation was a double-blind crossover study, which included a follow-up of 19 planters over a 3-week period. During Week 1, the 19 planters handled untreated conifer seedlings while they planted imidacloprid- and cypermethrin-treated seedlings during study Week 2 and 3, respectively. Signs and symptoms of acute health effects were documented by a questionnaire, administered by the field staff, during these 3 weeks. Inflammation markers in the nasal mucous membrane were also measured as an objective test. Exposure to cypermethrin was further assessed by measuring 3-phenoxybenzoic acid (3-PBA) in urine. No validated biomarker was available to assess internal exposure to imidacloprid. RESULTS: No clear, acute adverse health effects could be found in planters during the week of exposure to conifer seedlings treated with imidacloprid (Merit Forest) or cypermethrin (Forester), as compared to during the week of planting untreated seedlings. During the week of cypermethrin exposure, the individuals had 3-PBA values that were 12-54% higher (P < 0.05), depending on the worker, than those observed during the untreated week. There were no statistically significant correlations between the raised levels of 3-PBA and self-reported health problems. These results have been obtained during planting in late summer/early autumn and with good use of protective clothing. CONCLUSIONS: No clear, acute adverse health effects could be found in planters after exposure to conifer seedlings treated with imidacloprid (Merit Forest) or cypermethrin (Forester), as compared with planting untreated seedlings. The metabolite, 3-PBA, was found in low levels in urine and was increased after exposure to cypermethrin. However, no clear relationships could be found between exposure and reported symptoms or between elevated 3-PBA levels and reported symptoms.

Integrated microfluidic system for electrochemical sensing of urinary proteins.

This study reports a new microfluidic system integrated with a microfluidic control module and a micro electrochemical module for detection of urinary proteins. The integrated microsystem can automatically detect proteins in urine with a high sensitivity. The microfluidic control module consists of a new two-way, spiral-shape micropump which can transport the urine samples to the sensing regions. The net ionic charges of the protein samples can be detected while the samples flow through the sensing region of the micro electrochemical module. Two major urinary proteins including lysozyme and albumin are detected in a multiple-channel layout with little human intervention and are analyzed in a short period of time, while only consuming a 100-mul urine sample. The developed microfluidic system could lead to a convenient, yet crucial, platform for chemical and biological detection and diagnosis.

Modulation of the transforming growth factor beta1 by vitamin E in early nephropathy.

OBJECTIVE: The aim of this study was to investigate the pharmacological activity of an antioxidant, alpha-tocopherol (vitamin E, VE) in streptozotocin-induced diabetic rats and study its role in modulating the transforming growth factor beta1 (TGF-beta1). METHODS: Male Sprague-Dawley rats were treated with streptozotocin to induce diabetes. VE and/or insulin (INS) were administered daily during treatment periods of 3, 5, 7 and 10 days. Plasma glucose and fructosamine were measured in diabetic rats at the end of each treatment period. Samples of plasma, urine and renal cortex were analyzed for changes in protein and lysozyme excretion, reduced glutathione and malondialdehyde formation. TGF-beta1 was determined by ELISA and expression of TGF-beta1 mRNA was investigated by RT-PCR and Northern blot analysis. RESULTS: Diabetes-induced glycemic stress was suppressed by INS, VE or a combination of INS and VE. Diabetes-induced increases of glucose, protein and lysozyme excretion were markedly depressed after 10-day treatment with INS, VE and the combination of INS and VE. Decreased glutathione content in the renal cortex of diabetic rats recovered towards control values, especially after 10-day treatment. Malondialdehyde content increased in diabetic rats and was reduced towards control value following 7- and 10-day treatments. Treatment of diabetic rats with INS, VE or the combination of INS and VE decreased elevated TGF-beta1 in plasma, decreased excretion of TGF-beta1 in urine, and decreased renal cortex TGF-beta1 mRNA levels. CONCLUSIONS: Diabetes-induced overexpression of TGF-beta1 mRNA was suppressed by VE and INS after 5-, 7- and 10-day treatments. The results obtained with the antioxidant VE suggest that oxidative stress is involved in the development of diabetic nephropathy. Therefore, VE treatment may be effective in early stages of diabetic nephropathy to decrease or prevent pathological complications.

Lysosomal enzymes in preeclamptic women in northern Nigeria.

BACKGROUND: The incidence of preeclampsia is high in northern Nigeria, as it is in many other developing countries, and preeclampsia is associated with significant maternal and fetal morbidity and mortality. We inquired if proteinuria or hypertension alone could account for the altered concentrations of urinary lysosomal hydrolases that have been reported in preeclamptic women and pregnant women without preeclampsia. METHODS: The activities of urinary beta-hexosaminidase and beta-galactosidase were determined fluorometrically in pregnant women assigned to one of four groups: Group I: 41 preeclamptic women; Group II: 31 hypertensive aproteinuric women; Group III: 44 normotensive proteinuric women; and Group IV: 52 healthy pregnant women (controls). RESULTS: The urinary beta-hexosaminidase concentrations were decreased in the preeclamptic women (P<0.005) and proteinuric women (P<0.001) when compared to the healthy pregnant controls. There was no significant difference in beta-hexosaminidase concentrations between the hypertensive women and the healthy pregnant controls. The urinary beta-galactosidase concentrations for preeclamptic, hypertensive, and proteinuric women did not differ significantly versus healthy pregnant controls. CONCLUSIONS: The reduced urinary excretion of beta-hexosaminidase in preeclamptic women is associated with proteinuria, but not hypertension. Measuring urinary concentrations of lysosomal hydrolases alone or in conjunction with urinary protein concentrations is not likely to be useful in predicting or monitoring the clinical course of preeclampsia; however, it might prove important in gaining a more complete understanding of the pathogenesis of renal tubular epithelial cell injury and proteinuria that occurs in preeclampsia.

Laparoscopic right nephrectomy for live kidney donation: functional results.

Laparoscopic live-donor nephrectomy has gained wide acceptance. However, the vast majority of surgeons perform left nephrectomies only, which may not always be in the best interest of the donor. Of 17 consecutive laparoscopic donor nephrectomies, 13 were done on the right side. The function of these grafts was compared with that of 17 kidneys previously procured by an open technique and with that of the four left laparoscopic grafts. Ischaemic damage was evaluated by post-operative nuclear scanning and urinary lysozyme, and graft function by creatinine and creatinine clearance. Results show that operating time was longer in the laparoscopic donors, but identical in right and left laparoscopic procurements. Ischaemic damage and function were similar, regardless of the side or the surgical technique. We can conclude that right laparoscopic donor nephrectomy is feasible and results in good graft function. Systematic harvesting from the left side may, therefore, not be justified.