Ultrasensitive haptoglobin biomarker detection based on amplified chemiluminescence of magnetite nanoparticles.

BACKGROUND: Haptoglobin is an acute-phase protein used as predicting diagnostic biomarker both in humans (i.e., diabetes, ovarian cancer, some neurological and cardiovascular disorders) and in animals (e.g., bovine mastitis). The latter is a frequent disease of dairy industry with staggering economical losses upon decreased milk production and increased health care costs. Early stage diagnosis of the associated diseases or inflammation onset is almost impossible by conventional analytical manners. RESULTS: The present study demonstrates a simple, rapid, and cost-effective label-free chemiluminescence bioassay based on magnetite nanoparticles (MNPs) for sensitive detection of haptoglobin by employing the specific interaction of hemoglobin-modified MNPs. The resulting haptoglobin-hemoglobin complex inhibits the peroxidase-like activity of luminol/H2O2-hemoglobin-MNPs sensing scheme and reduces the chemiluminescence intensities correspondingly to the innate haptoglobin concentrations. Quantitative detection of bovine haptoglobin was obtained within the range of 1 pg mL-1 to 1 µg mL-1, while presenting 0.89 pg mL-1 limit of detection. Moreover, the influence of causative pathogenic bacteria (i.e., Streptococcus dysgalactiae and Escherichia coli) and somatic cell counts (depicting healthy, sub-clinical and clinical mastitis) on the emitted chemiluminescence radiation were established. The presented bioassay quantitative performances correspond with a standardized assay kit in differentiating dissimilar milk qualities. CONCLUSIONS: Overall, the main advantage of the presented sensing concept is the ability to detect haptoglobin, at clinically relevant concentrations within real milk samples for early bio-diagnostic detection of mastitis and hence adjusting the precise treatment, potentially initiating a positive influence on animals' individual health and hence on dairy farms economy.

Quick colorimetric determination of choline in milk and serum based on the use of MoS2 nanosheets as a highly active enzyme mimetic.

The authors have synthesized molybdenum disulfide nanosheets (MoS2 nanosheets) by using a bottom-up hydrothermal method. The nanosheets display strong catalytic (enzyme mimetic) activity in catalyzing the oxidation of peroxidase substrate of 3,3',5,5'-tetramethylbenzidine (TMB) in presence of H2O2 to produce a blue product. The peroxidase mimicking properties of MoS2 nanosheets depend on temperature, H2O2 concentration and pH value. A choline assay was worked out where choline was oxidized by choline oxidase in presence of oxygen to produce H2O2 which is colorimetrically detected, best at 652 nm. The method works in the 1 to 180 µM choline concentration range with a 0.4 µM detection limit. Color changes may also be detected visually. The assay is simple, highly sensitive, selective and rapid. It was applied in the determination of choline in (spiked) milk and serum. Graphical abstract Basic principle of intrinsic peroxidase-like activity of MoS2 nanosheets, applied to design a rapid and selective colorimetric assay for choline detection based on the tetramethylbenzidine (TMB) color reaction.

A nanoporous palladium(II) bridged coordination polymer acting as a peroxidase mimic in a method for visual detection of glucose in tear and saliva.

A nanoporous coordination polymer (NPCP) was prepared from palladium(II) chloride and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole and is shown to act as a peroxidase mimetic. It can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 which is formed on enzymatic oxidation of glucose by glucose oxidase. Based on these findings, a sensitive glucose test was worked out at 652 nm where the intensity if the greenish-blue product is related to the actual concentration of glucose. Figures of merit include (a) rather low Km value (30 µM) which evidences the strong binding affinity of the NPCP toward glucose, (b) a high v(max) (8.5 M·s-1), (c) a 47 nM detection limit, (d) a lifetime of a month, (e) a wide working pH range (2-10), and (f) a 25-80 °C temperature range. The assay was applied to non-invasive determination of glucose assay in tear, saliva where the detection limits are found to be 61 and 91 nM, respectively. Graphical abstract DSchematic of the mechanism of the peroxidase like catalytic activity of AHMT-Pd NPCP that was applied in a selective colorimetric method for glucose detection based on TMB oxidation in the presence of enzymatically generated H2O2.

Homogenous Dispersion of MoS2 Nanosheets in Polyindole Matrix at Air-Water Interface Assisted by Langmuir Technique.

Two-dimensional (2D) inorganic layered materials when embedded in organic polymer matrix exhibit exotic properties that are grabbing contemporary attention for various applications. Here, nanosheet morphology of molybdenum disufide (MoS2) synthesized via one-pot facile hydrothermal reaction are exfoliated in benign aqueous medium in the presence of indole to obtain a stable dispersion. These exfoliated nanosheets then act as host to template the controlled polymerization of indole. The preassembled MoS2-polyindole (MoS2-PIn) nanostructures are reorganized at the air-water interface using the Langmuir method to facilitate maximum interfacial interaction between nanosheet and polymer. This report emphasizes large area, homogeneous dispersion of uniform-sized MoS2 nanosheets (40-60 nm diameter) in the PIn matrix and the formation of stable and uniform film via the Langmuir-Schaefer (LS) method. These self-assembled, MoS2 decorated PIn LS films are characterized using atomic force microscopy (AFM) and transmission electron microscopy (TEM). The fabricated LS films in sandwiched structure Al/MoS2-PIn/ITO as the Schottky diode portrayed remarkable enhancements in charge transport properties. Our study illustrates the potential of the MoS2-PIn LS film in electronic applications and opens a new dimension for uniform dispersion of 2D materials in other polymers via the Langmuir method for device fabrication and enhancement of electrical properties.

N-acetyl-ß-d-glucosaminidase activity assay for monitoring insulin-dependent diabetes using Ag-porous Si SERS platform.

Determination of urinary or serum N-acetyl-ß-d-glucosaminidase (NAG) activity as a tissue damage indicator is widely used in diagnosis of various pathologies, including diabetic nephropathy. Early and rapid biomarker detection is an important element of medical diagnosis, facilitating prompt therapeutic decisions and prognosis evaluation. Herein, we present a modified sensing approach for a rapid and reliable NAG activity determination in complex media using surface-enhanced Raman spectroscopy (SERS). Porous silicon (PSi) Fabry-Pérot interferometers were redesigned as sensitive SERS platforms utilizing the vast inherent surface area for silver (Ag) nanoparticles embedment. Interaction of the porous nanostructures with specific NAG-enzymatic products produces an indicative spectral fingerprint proportional in magnitude to its concentration. The sensitivity of Ag-PSi SERS substrates was evaluated in complex matrices presenting sufficient limits of detection compared with other advanced assays and techniques (0.07, 0.47 and 0.50 mU mL-1 for urine, milk and plasma, respectively). The augmented optical performance revealed recovery values of 96-109%, indicating successful and selective NAG recognition in biological fluids. Finally, the potential applicability of the suggested prototype for real-life scenarios was evaluated in vivo, in a model of insulin-dependent diabetes induced in sheep. Overall, the robust data confirm the application of SERS analysis for early diagnosis of pathology and for evaluation of clinical responses to pharmacological treatments.

Bovine mastitis inflammatory assessment using silica coated ZnO-NPs induced fluorescence of NAGase biomarker assay.

Bovine mastitis (BM) is the most common inflammatory disease in the dairy sector worldwide, originated from bacterial invasion onto the mammary gland. Early BM detection is crucial for identifying new pathogenic infections within the dairy herd, which can be alleviated by antimicrobial therapy. N-acetyl-ß-D-glucosaminidase (NAGase) is a prominent BM inflammatory biomarker secreted onto the blood circulation upon pathogenesis and then released into milk, capable of separating healthy quarters from subclinical and clinical BM cases. Herein, we report on a sensitive differentiation assay of BM severity based on enhanced fluorescence emission of a conventional NAGase activity assay. The addition of silica-coated zinc oxide nanoparticles induces non-radiative energy transfer to the lysosomal reaction products, thus leading to enhanced fluorescence (above 3-fold). Various milk qualities within the entire inflammatory spectrum were evaluated by the modified fluorescence assay with respect to non-infected milk. The amplified emission values differentiate between two predominant BM causative pathogens (Streptococcus dysgalactiae and Escherichia coli) at various somatic cell counts. In general, the presented concept offers an efficient, simple, cost-effective fluorescence signal augmentation for mastitis identification, thus offering means to diagnose the severity of the associated disease.

Amplified Fluorescence by ZnO Nanoparticles vs. Quantum Dots for Bovine Mastitis Acute Phase Response Evaluation in Milk.

Bovine mastitis (BM) is a prominent inflammatory disease affecting the dairy industry worldwide, originated by pathogenic agent invasion onto the mammary gland. The early detection of new BM cases is of high importance for infection control within the herd. During inflammation, various biomarkers are released into the blood circulation, which are consequently found in milk. Herein, the lysosomal activity of N-acetyl-ß-D-glucosaminidase (NAGase), a predominant BM indicator, was utilized for highly sensitive clinical state differentiation. The latter is achieved by the precise addition of tetraethyl orthosilicate-coated zinc oxide nanostructures (quantum dots or nanoparticles, individually) onto a conventional assay. Enhanced fluorescence due to the nanomaterial accumulative near-field effect is achieved within real milk samples, contaminated with Streptococcus dysgalactiae, favoring quantum dots over nanoparticles (> 7-fold and 3-fold, respectively), thus revealing significant differentiation between various somatic cell counts. The main advantage of the presented sensing concept, besides its clinically relevant concentrations, is the early bio-diagnostic detection of mastitis (subclinical BM) by using a simple and cost-effective experimental setup. Moreover, the assay can be adapted for BM recovery prognosis evaluation, and thus impact on udder health status, producing an alternative means for conventional diagnosis practices.

Gold Nanoparticle Size-Dependent Enhanced Chemiluminescence for Ultra-Sensitive Haptoglobin Biomarker Detection.

Bovine mastitis (BM) is a frequent disease in the dairy industry that causes staggering economical losses due to decreased milk production and increased health care costs. Traditionally, BM detection depends on the efficacy and reliability of analytical techniques that measure somatic cell counts (SCC), detect pathogens, and reveal inflammatory status. Herein, we demonstrate the detection of bovine haptoglobin, a well-documented acute phase protein for evaluating BM clinical status, by utilizing hemoglobin-binding capacity within luminol chemiluminescence (CL) system. The resulting haptoglobin-hemoglobin complex reduces the CL signal proportionally to inherent haptoglobin concentrations. Different sizes of cross-linked gold nanoparticles (GNPs) were examined for enhanced CL (eCL) signal amplification, presenting over 30-fold emitted radiation enhancement for optimized size within real milk samples with respect to nanoparticle-free assay. The eCL values were proportionally related to nanoparticle size and content, influenced by SCC and pathogen type (e.g., Escherichia coli and coagulase-negative staphylococci). The optimized bioassay showed a broad linear response (1 pg mL-1-10 µg mL-1) and minute detection limit of 0.19 pg mL-1, while presenting quantitative performance in agreement with commercial ELISA kit. Finally, the resulting optimized eCL concept offers an efficient label-free detection of haptoglobin biomarker, offering means to diagnose the severity of the associated diseases.

Enhanced Fluorescence of N-Acetyl-ß-D-Glucosaminidase Activity by ZnO Quantum Dots for Early Stage Mastitis Evaluation.

Recurrent mastitis events are the major cause of annual revenue losses in the dairy sector resulting in decreased milk yield, escalading treatment costs and increased health risk of the entire herd. Upon udder inflammation, several biomarkers are proportionally secreted to its severity onto the blood circulation and consequently into milk (upon breached blood-milk barrier). N-acetyl-ß-D-glucosaminidase activity is widely used mastitis indicator in milk, offering simple means of differentiation between healthy quarters from those with subclinical or clinical severity. Herein, we demonstrate fluorescence signal amplification concept for sensitive clinical status discrimination. Tetraethyl orthosilicate coated zinc oxide quantum dots were employed within the conventional N-acetyl-ß-D-glucosaminidase activity assay. Under the experimental conditions, a profound non-radiative energy transfer occurred between quantum nanomaterials onto enzymatic fluorescent products resulting in intensified emission of the latter, over 11-folds, in comparison to nanoparticle-free assay. Overall, the fluorescence intensities were proportionally related to zinc oxide quantum dots surface coverage and concentration, SCC values and influenced by the causing bacteria (i.e., Streptococcus dysgalactiae and Coagulase-negative Staphylococci). Finally, the presented proof-of-concept offers an efficient, simple, cost-effective fluorescence signal amplification for early stage mastitis identification, offering means to diagnose the severity of the associated diseases and hence deducing on animals' clinical status.

Milk haptoglobin detection based on enhanced chemiluminescence of gold nanoparticles.

The suggested research specifically addresses the major source of economic loss of the dairy industry, the bovine mastitis (BM), an inflammatory disease of mammary gland caused by bacterial intramammary infection. During udder inflammation, the concentrations of acute phase proteins (APP) in both plasma and milk are escalated, which can be distinctively utilized as predicting diagnostic biomarkers of cattle's BM clinical status. Herein, we demonstrate a liquid-phase luminol chemiluminescence (CL) system for sensitive detection of haptoglobin (Hp), a predictive APP of BM, by utilizing the binding capacity of hemoglobin (Hb). The CL intensity is linearly proportional to Hb-Hp complex formation, resulting in peroxidase-like activity inhibition of luminol-H2O2-Hb CL system. Enhanced CL, at least 10-fold effect within real samples, is attained by the addition of catalytically active cross-linked gold nanoparticles (GNPs) onto the luminol-H2O2 solution. Moreover, the influence of different somatic cell counts (representing subclinical and clinical BM status) and pathogen types (i.e., CNS and Streptococcus dysgalactiae) on the secreted milk Hp levels obtained from Holstein cows are established. The analyzed Hp concentrations are in agreement with a commercial enzyme-linked immunosorbent assay kit. The proposed CL sensing concept offers cost-effective, simple, label-free and reliable systematic analysis of Hp biomarker for BM, potentially initiating a positive effect on animals' health and overall economy of the dairy farms.

Colorimetric detection of cholesterol based on enzyme modified gold nanoparticles.

We develop a simple colorimetric method for determination of free cholesterol in aqueous solution based on functionalized gold nanoparticles with cholesterol oxidase. Functionalized gold nanoparticles interact with free cholesterol to produce H2O2 in proportion to the level of cholesterol visually is being detected. The quenching in optical properties and agglomeration of functionalized gold nanoparticles play a key role in cholesterol sensing due to the electron accepting property of H2O2. While the lower ranges of cholesterol (lower detection limit i.e. 0.2mg/dL) can be effectively detected using fluorescence study, the absorption study attests evident visual color change which becomes effective for detection of higher ranges of cholesterol (lower detection limit i.e. 19mg/dL). The shades of red gradually change to blue/purple as the level of cholesterol detected (as evident at 100mg/dL) using unaided eye without the use of expensive instruments. The potential of the proposed method to be applied in the field is shown by the proposed cholesterol measuring color wheel.

A comparative Study of Aptasensor Vs Immunosensor for Label-Free PSA Cancer Detection on GQDs-AuNRs Modified Screen-Printed Electrodes.

Label-free and sensitive detection of PSA (Prostate Specific Antigen) is still a big challenge in the arena of prostate cancer diagnosis in males. We present a comparative study for label-free PSA aptasensor and PSA immunosensor for the PSA-specific monoclonal antibody, based on graphene quantum dots-gold nanorods (GQDs-AuNRs) modified screen-printed electrodes. GQDs-AuNRs composite has been synthesized and used as an electro-active material, which shows fast electron transfer and catalytic property. Aptamer or anti-PSA has immobilized onto the surface of modified screen printed electrodes. Three techniques are used simultaneously, viz. cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedence spectroscopy (EIS) to investigate the analytical performance of both PSA aptasensor and PSA immunosensor with its corresponding PSA antigen. Under optimum conditions, both sensors show comparable results with an almost same limit of detection (LOD) of 0.14 ng mL-1. The results developed with aptasensor and anti-PSA is also checked through the detection of PSA in real samples with acceptable results. Our study suggests some advantages of aptasensor in terms of better stability, simplicity and cost effectiveness. Further our present work shows enormous potential of our developed sensors for real application using voltammetric and EIS techniques simultaneous to get reliable detection of the disease.

One step electro-oxidative preparation of graphene quantum dots from wood charcoal as a peroxidase mimetic.

In present study, we highlight one-step electrochemical synthesis of nearly uniform size (~ 5nm) of graphene quantum dots (E-GQDs) from wood charcoal and their further application as a peroxidase enzyme mimetic. The structural and optical properties of as-synthesized E-GQDs were probed by TEM, AFM, and spectroscopic techniques. Peroxidase enzyme mimetic potential of E-GQDs were examined for colorimetric detection of H2O2 and glucose. E-GQDs allowed a rapid and sensitive detection of glucose with a detection limit of 0.006mM for dynamic response range of 0.01-0.6mM. The calculated higher value of Vmax (7.2 × 10-7Ms-1) along with lower Km (0.012mM) corroborate enhanced the peroxidase-like activity of E-GQDs. Study introduces a cheap and widely available raw material for the electrochemical synthesis of graphene quantum dots with commendable enzyme mimetic activity which may have a huge impact in developing calorimetric bioanalysis systems.

Urease immobilized fluorescent gold nanoparticles for urea sensing.

We report a surfactant-free synthesis of monodispersed gold nanoparticles (AuNPs) with average size of 15 nm. An approach for visual and fluorescent sensing of urea in aqueous solution based on shift in surface plasmon band (SPB) maxima as well as quench in fluorescence intensity. To enable the urea detection, we functionalized the thiol-capped gold nanoparticles with urease, the enzyme specific to urea using carbodiimide chemistry. The visible color changed of the gold colloidal solution from red to blue (or purple); this was evident from quenching in absorbance and fluorescence intensity, is the principle applied here for the sensing of urea. The solution turns blue when the urea concentration exceeds 8 mg/dL which reveals visual lower detection limit. The lower detection limits governed by the fluorescence quenching were found 5 mg/dL (R(2) = 0.99) which is highly sensitive and selective compared to shift in SPB maxima. The approach depicted here seems to be important in clinical diagnosis.

Different shades of cholesterol: Gold nanoparticles supported on MoS2 nanoribbons for enhanced colorimetric sensing of free cholesterol.

In the present study, we manifest that traditionally used gold nanoparticles when supported on molybdenum disulfide nanoribbons matrix (MoS2 NRs-Au NPs) show synergistically enhanced intrinsic peroxidase like catalytic activity and can catalyze the oxidation of 3,3',5,5' tetramethyl benzidine by H2O2 to produce a highly sensitive blue shade product depending on level of free cholesterol, when tested on complex system of human serum. Further the system attests appreciable kinetics, owing to Km value as low as 0.015 mM and better loading capacity (Vmax=6.7×10(-6) M s(-1)). Additionally, the proposed system is stable for weeks with ability to perform appreciably in wide pH (3-6) and temperature range (25-60 °C). Utilizing this potential, the present work proposes a cholesterol detection color wheel which is used along with cost effective cholesterol detection strips fabricated out of proposed MoS2 NRs-Au NPs system for quick and reliable detection of free cholesterol using unaided eye.

Partially reduced graphene oxide-gold nanorods composite based bioelectrode of improved sensing performance.

The present work proposes partially reduced graphene oxide-gold nanorods supported by chitosan (CH-prGO-AuNRs) as a potential bioelectrode material for enhanced glucose sensing. Developed on ITO substrate by immobilizing glucose oxidase on CH-prGO-AuNRs composite, these CH-prGO-AuNRs/ITO bioelectrodes demonstrate high sensitivity of 3.2 µA/(mg/dL)/cm(2) and linear range of 25-200 mg/dL with an ability to detect as low as 14.5 mg/dL. Further, these CH-prGO-AuNRs/ITO based electrodes attest synergistiacally enhanced sensing properties when compared to simple graphene oxide based CH-GO/ITO electrode. This is evident from one order higher electron transfer rate constant (Ks) value in case of CH-prGO-AuNRs modified electrode (12.4×10(-2) cm/s), in contrast to CH-GO/ITO electrode (6×10(-3) cm/s). Additionally, very low Km value [15.4 mg/dL(0.85 mM)] ensures better binding affinity of enzyme to substrate which is desirable for good biosensor stability and resistance to environmental interferences. Hence, with better loading capacity, kinetics and stability, the proposed CH-prGO-AuNRs composite shows tremendous potential to detect several bio-analytes in the coming future.