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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.

A graphene analogous of transition metal dichalcogenides, mainly molybdenum disulfide (MoS2), have recently attracted wide attention due to its catalytic and electrical properties. Herein, we report a simple one step synthesis of gold nanoparticles (AuNPs) decorated over MoS2 quantum dots (AuNPs@MoS2-QDs) as a robust peroxidase- mimetic material. The AuNPs@MoS2-QDs composite catalyzes the peroxidase substrate of 3, 3′, 5,5′-tetramethylbenzidine (TMB) oxidation in the presence of H2O2 to produce a bluish-green colored product. The peroxidase mimetic behavior of AuNPs@MoS2-QDs composite complies with the steady state kinetics and is stable in harsh conditions such as high or low temperature and pH. The developed composite system has been used for sensitive and selective detection of glucose. The developed biosensor shows an excellent detection in dynamic ranges from 1 to 400 μM (R2∼0.996) with the detection limit of 0.068 μM in PBS buffer solution. Low Km value (0.06 mM) of AuNPs@MoS2-QDs has shown strong binding affinity towards substrates along with high values of Vmax (10.6 × 10−6 Ms−1) indicating greater catalytic reaction rate. Further the biosensor is developed as a portable test kit for detection of glucose in biological fluids like serum, tear and saliva utilizing agarose hydrogel as a visual detection platform. Our portable sensing kit shows an excellent response for detection of glucose in real samples compared to the conventional methods.

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.

Bovine mammary gland is susceptible to numerous bacterial infections resulting in inflammatory disease condition of bovine mastitis (BM) with a staggering economic impact on the dairy industry worldwide. Early BM detection is crucial for infection control within the dairy herd, which can be alleviated by antimicrobial therapy. N-acetyl-β-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 a sensitive, cost-effective and handy-to-use BM severity assay based on NAGase inherent content found in whole milk samples. Silver-coated porous Si (Ag-PSi) Fabry–Perot interferometers were employed for quantifying the lysosomal activity in different milk qualities using surface-enhanced Raman spectroscopy (SERS). The optical response was proportional to the occurring pathogenesis induced by predominant bacteria found in dairy farms at different somatic cell levels. The optimized Ag-PSi SERS substrates were utilized for quantitative analysis of NAGase levels found at clinically relevant BM concentrations while presenting the detection limit of 0.27 μM min−1. The optical performances were associated with the conventional standardized approach in terms of recovery values (85–98%), thus presenting sufficient adaptability to complex matrix analysis as whole milk.

In the current work, we have fabricated a paper test strip for visual assay of choline by using choline oxidase enzyme with H2O2 based on the oxidation product of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) catalyzed by gold nanoparticles decorated WS2 quantum dots (AuNPs-WS2QDs) to appear blue color product. Under a large range of temperature, pH and H2O2 concentration, AuNps-WS2QDs exhibit prominent peroxidase-like property. In the presence of oxygen, choline oxidized by choline oxidase which produces H2O2 is colorimetrically measured, at 652 nm. The blue color appears due to biochemical reactions between TMB and AuNps-WS2QDs were used as an indication for estimation of H2O2. The proposed scheme is applied for choline detection in concentration range of 1–150 μM in PBS with the 0.086 µM limit of detection. Color variation can be observed visually and this assay is selective, simple and quick. Further portable test kit was fabricated for choline detection in real samples i.e. serum and milk which shows an excellent response towards choline detection. The filter paper test strip changed color intensity based on the concentration of choline.

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-β-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.

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 transmi...

MoS2 quantum dots were hydrothermally synthesized and utilized for the formation and stabilization of a nanocomposite with silver nanoparticles (AgNPs) in a single step. This composite was characterized by transmission electron microscopy and zeta potential measurements. It is found that this nanohybrid can be stimulated by mercury(II) ion and then exhibits excellent oxidase mimicking activity. The oxidase-like activity is demonstrated by the oxidation of 3,3′,5,5′-tetramethylbenzidine by H2O2 that leads to the formation of a blue product. An assay was developed for determination of cysteine (Cys) at ultra-trace level because Cys inhibits the activity of the nanozyme via interaction with Hg(II). The Cys assay, best performed at a wavelength of 652 nm, works in the 1–100 μM concentration range and has a 0.82 μM detection limit. In addition, a portable Cys test kit is described that was applied to the determination of Cys in serum samples. The resulting colorations were compared with color chat wheel. The method is simple, rapid, cost-effective, and sensitive.

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&ndash, 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&minus, 1&ndash, 10 &micro, g mL&minus, 1) and minute detection limit of 0.19 pg mL&minus, 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.

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.

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.

The authors have synthesized molybdenum disulfide nanosheets (MoS

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.

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.

The present work introduces partially reduced graphene oxide (pRGO)-silica (SiO2) particles hybrid system (pRGOSHs) for sensitive and cost effective free cholesterol detection. Fabricated out of thin layers of pRGOSHs, these proposed ChOx/pRGOSHs/ITO based biosensors have a detection range of 2.6–15.5 mM with an appreciable detection limit of 1.3 mM and sensitivity of 11.1 μA/mM/cm2. Low Michaelis–Menten constant (Km) (4.9 × 10− 4 mM) and high diffusivity constant (D) (3.2 × 10− 10 cm2/s) values clearly indicate enhanced immobilization of enzyme over the substrate. Additionally, electrochemical impedance studies indicate that the synergistic combination of SiO2 and pRGO also results in much lower impedance values (40% and 18% decrease in comparison to SiO2 and pRGO respectively) for an overall enhanced sensing performance. These results are further corroborated by the density functional theory based theoretical simulations indicating enhanced electron density (theoretically) in case of the proposed hybrid system. Finally, the present work also highlights the importance of Si–OH bonds formation in the proposed pRGOSHs composite system for attaining such enhanced biosensing ability.

In the present study, we report graphene quantum dots (GQDs), an enzyme mimetic of horse radish peroxidase (HRP), for unprecedented detection of free cholesterol. Synthesized directly from graphite using simple and quick one step wet chemical method, these GQDs in the presence of H 2 O 2 exhibit highly efficient catalytic activity toward the oxidation of peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) to produce a blue colored product. The proposed detection system based on GQDs allows wide range (0.02–0.6 mM) of cholesterol sensing with a detection limit as low as 0.006 mM. Further, higher V max (7.3 × 10 −6 M s −1 ) along with lower K m (0.01 mM) attest enhanced peroxidase like catalytic activity and better binding affinity of cholesterol oxidase (ChOx) to cholesterol resulting in good biosensor stability and resistance to environmental interferences. The proposed method without the use of sophisticated instruments perceives the cholesterol using naked eye with blue color compound formation. The potential of the method to be applied on field is shown by the proposed cholesterol measuring color wheel, where the shades of color are related to actual levels of cholesterol in the sample.

Realizing the unavailability of fast and reliable diagnostic techniques, especially for cholesterol measurement, the present work reports the development of cost effective bioelectrodes based on a reduced graphene oxide–functionalized gold nanoparticle (∼25 nm) hybrid system (RGO–Fn Au NPs). The electrodes fabricated by the electrophoretic deposition technique attest a synergistically enhanced electrochemical sensing ability of 193.4 μA mM−1 cm−2 for free cholesterol detection, which is much higher than that of the traditional RGO system. The electrochemical impedance studies (EIS) show low charge transfer resistance, RCT, for the hybrid system which is 57% and 60% lower than those of RGO and Au NPs respectively. Also higher loading capacity and enhanced kinetics have been realized for the hybrid system, owing to lower Km value (0.005 mM) and enhanced rate constant (3.8 × 10−4 cm s−1) in comparison with RGO and Au NPs. Moreover, the RGO–Fn Au NP platform promises a wider range of cholesterol detection (0.65–12.93 mM), while simultaneously being capable of detecting as low as 0.34 mM of free cholesterol. Apart from better sensitivity, loading capacity, kinetics and detection range, the system also has appreciable selectivity and stability. This supports its potential to be brought on field in the near future for cost effective and reliable detection from the complex system of human serum.

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 (MoS

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.

A polyaniline–gold (PAni–Au) nanocomposite is chemically synthesized and impregnated in a chitosan matrix for immobilization of cholesterol oxidase on an indium tin oxide-coated glass plate for development of cholesterol biosensors. The PAni–Au nanocomposite is characterized for structural and thermal properties. Further the PAni–Au nanocomposite is used for cholesterol oxidase immobilization and linkage is examined by FTIR. The nanocomposite is used to form a modified bioelectrode by immobilizing cholesterol oxidase in the chitosan matrix and examined under an optical microscope for uniformity and morphological studies. A modified bioelectrode (ChOx/PAni–Au–CH/ITO) is used to detect cholesterol by using a voltammetric technique with a redox mediator. The sensor exhibits linearity in a wide range of 50–500 mg dL−1 with a detection limit of 37.89 mg dL−1, a sensitivity of 0.86 μA mg dL−1 and a shelf life of more than 3 weeks when stored at 4 °C. Voltammetric studies have also been carried out with common possible interferents. Responses are recorded to get enzyme–substrate kinetics for the biosensor and found as Km = 10.84 mg dL−1. The low value of Km indicates that the prepared nanocomposite facilitates the enzymatic reaction and enzymatic activity. Excellent immobilization and enzyme–substrate reactions show a distinct advantage of this matrix over other matrixes used for cholesterol biosensors. The novelty of the prepared electrode lies in its reusability, higher sensitivity, better shelf life, and accuracy.

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.

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.