Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine.

The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an o-phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Discrimination of Staphylococcus aureus Strains from Coagulase-Negative Staphylococci and Other Pathogens by Fourier Transform Infrared Spectroscopy.

Staphylococcus aureus is an extremely infectious and malignant pathogen among many bacteria species. The aim of this work is to provide a robust classification model that would be able to identify S. aureus independent of the culture growth stage and the variations in bacteria concentration in suspension and also one that would be able to identify the pathogen among both taxonomically close species of the same genus and taxonomically distant species of different genera, using Fourier transform infrared spectroscopy (FTIR). In total, the spectra of 141 isolates of 17 bacteria have been used. Based on a combination of principal component analysis (PCA) and linear discriminant analysis (LDA), an identification model providing 100% sensitivity and 98% specificity was built. Inherent reliability and flexibility of the model have been shown. The proposed method of analysis allows us to get closer to the diagnostic requirements in the field of clinical microbiology, and it can be utilized for typing of other pathogenic bacteria species.

Surface-enhanced Raman spectroscopy in tandem with a gradient electric field from 4-mercaptophenylboronic acid on silver nanoparticles.

The surface-enhanced Raman spectroscopy (SERS) signal of a reporter on silver nanoparticles can be effectively gained by gradient electric field application. The external electric field initiates the dielectrophoresis of nanoparticles and their electrically induced dipole-dipole interaction. Owing to dielectrophoresis, the nanoparticles are concentrated in the area of high electrical field strength. The induced dipole-dipole interaction leads to additional coagulation of nanoparticles and formation of hotspots. Both dielectrophoresis and induced dipole-dipole interaction increase the number of hotspots, which leads to a SERS signal growth. These two mechanisms of SERS signal amplification are explained by the dielectrophoresis and Derjaguin-Landau-Verwey-Overbeek theories. The benefits of the surface-enhanced Raman spectroscopy in tandem with the gradient electric field are experimentally confirmed using a SERS-active reporter, 4-mercaptophenylboronic acid which has a characteristic peak at Raman shift of 1586 cm-1, conjugated to silver nanoparticles of 32, 52, 58, and 74 nm in diameter. The SERS signal gain depends on the silver nanoparticle stability, size, and electric field strength. The limit of detection for 4-mPBA in the system under study can be calculated from the concentration plot and equals to 63 nM. The enhancement factor calculated for SERS in tandem with the gradient electric field can reach 106.Graphical abstract.

Cryostructuring of Polymeric Systems †: Application of Deep Neural Networks for the Classification of Structural Features Peculiar to Macroporous Poly(vinyl alcohol) Cryogels Prepared without and with the Additives of Chaotropes or Kosmotropes.

Macroporous poly(vinyl alcohol) cryogels (PVACGs) are physical gels formed via cryogenic processing of polymer solutions. The properties of PVACGs depend on many factors: the characteristics and concentration of PVA, the absence or presence of foreign solutes, and the freezing-thawing conditions. These factors also affect the macroporous morphology of PVACGs, their total porosity, pore size and size distribution, etc. In this respect, there is the problem with developing a scientifically-grounded classification of the morphological features inherent in various PVACGs. In this study PVA cryogels have been prepared at different temperatures when the initial polymer solutions contained chaotropic or kosmotropic additives. After the completion of gelation, the rigidity and heat endurance of the resultant PVACGs were evaluated, and their macroporous structure was investigated using optical microscopy. The images obtained were treated mathematically, and deep neural networks were used for the classification of these images. Training and test sets were used for their classification. The results of this classification for the specific deep neural network architecture are presented, and the morphometric parameters of the macroporous structure are discussed. It was found that deep neural networks allow us to reliably classify the type of additive or its absence when using a combined dataset.

Easy-Preparable Butyrylcholinesterase/Microgel Construct for Facilitated Organophosphate Biosensing.

A versatile guest matrix was fabricated from a temperature- and pH-sensitive poly(N-isopropylacrylamide)-co-(3-(N,N-dimethylamino)propylmethacrylamide) microgel (poly(NIPAM-co-DMAPMA), MG) for the gentle incorporation of butyrylcholinesterase (BChE). The microgel/BChE films were built up on a surface of graphite-based screen-printed electrodes (SPEs) premodified with MnO2 nanoparticles via a two-step sequential adsorption under careful temperature and pH control. On this basis, a rather simple amperometric biosensor construct was formed, which uses butyrylthiocholine as BChE substrate with subsequent MnO2-mediated thiocholine oxidation at a graphite-based SPE. The complexation of BChE with the microgel was found to be safe and effective, as confirmed by a high operational and rather good long-term storage stability of the resultant SPE-MnO2/MG/BChE biosensors. The small mesh size of the microgel with respect to the size of BChE results in a predominant outer complexation of BChE within the dangling chains of the microgel rather than a deep penetration of the enzyme into the microgels. Given such surface localization, BChE is easily accessible both for the substrate and for cholinesterase inhibitors. This was supported by the analytical characteristics of the SPE-MnO2/MG/BChE biosensor that were examined and optimized both for the substrate and for the enzyme detection. The SPE-MnO2/MG/BChE biosensor enabled precision detection of organophosphorus pesticides (diazinon(oxon), chlorpyrifos(oxon)) in aqueous samples with minimized interference from extraneous (nonanalyte) substances (e.g., ions of heavy metals). The detection limits for diazinon(oxon) and chlorpyrifos(oxon) were estimated to be as low as 6 × 10-12 M and 8 × 10-12 M, respectively, after 20 min of preincubation with these irreversible inhibitors of BChE.

Neuropathy target esterase in mouse whole blood as a biomarker of exposure to neuropathic organophosphorus compounds.

The adult hen is the standard animal model for testing organophosphorus (OP) compounds for organophosphorus compound-induced delayed neurotoxicity (OPIDN). Recently, we developed a mouse model for biochemical assessment of the neuropathic potential of OP compounds based on brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) inhibition. We carried out the present work to further develop the mouse model by testing the hypothesis that whole blood NTE inhibition could be used as a biochemical marker for exposure to neuropathic OP compounds. Because brain NTE and AChE inhibition are biomarkers of OPIDN and acute cholinergic toxicity, respectively, we compared NTE and AChE 20-min IC50 values as well as ED50 values 1 h after single intraperitoneal (i.p.) injections of increasing doses of two neuropathic OP compounds that differed in acute toxicity potency. We found good agreement between the brain and blood for in vitro sensitivity of each enzyme as well for the ratios IC50 (AChE)/IC50 (NTE). Both OP compounds inhibited AChE and NTE in the mouse brain and blood dose-dependently, and brain and blood inhibitions in vivo were well correlated for each enzyme. For both OP compounds, the ratio ED50 (AChE)/ED50 (NTE) in blood corresponded to that in the brain despite the somewhat higher sensitivity of blood enzymes. Thus, our results indicate that mouse blood NTE could serve as a biomarker of exposure to neuropathic OP compounds. Moreover, the data suggest that relative inhibition of blood NTE and AChE provide a way to assess the likelihood that OP compound exposure in a susceptible species would produce cholinergic and/or delayed neuropathic effects. Copyright © 2016 John Wiley & Sons, Ltd.

Engineering Systems with Spatially Separated Enzymes via Dual-Stimuli-Sensitive Properties of Microgels.

This work examines the adsorption regime and the properties of microgel/enzyme thin films deposited onto conductive graphite-based substrates. The films were formed via two-step sequential adsorption. A temperature- and pH-sensitive poly(N-isopropylacrylamide)-co-(3-(N,N-dimethylamino)propylmethacrylamide) microgel (poly(NIPAM-co-DMAPMA microgel) was adsorbed first, followed by its interaction with the enzymes, choline oxidase (ChO), butyrylcholinesterase (BChE), or mixtures thereof. By temperature-induced stimulating both (i) poly(NIPAM-co-DMAPMA) microgel adsorption at T > VPTT followed by short washing and drying and then (ii) enzyme loading at T < VPTT, we can effectively control the amount of the microgel adsorbed on a hydrophobic interface as well as the amount and the spatial localization of the enzyme interacted with the microgel film. Depending on the biomolecule size, enzyme molecules can (in the case for ChO) or cannot (in the case for BChE) penetrate into the microgel interior and be localized inside/outside the microgel particles. Different spatial localization, however, does not affect the specific enzymatic responses of ChO or BChE and does not prevent cascade enzymatic reaction involving both BChE and ChO as well. This was shown by the methods of electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and amperometric analysis of enzymatic responses of immobilized enzymes. Thus, a novel simple and fast strategy for physical entrapment of biomolecules by the polymeric matrix was proposed, which can be used for engineering systems with spatially separated enzymes of different types.

Dual-stimuli-sensitive microgels as a tool for stimulated spongelike adsorption of biomaterials for biosensor applications.

This work examines the fabrication regime and the properties of microgel and microgel/enzyme thin films adsorbed onto conductive substrates (graphite or gold). The films were formed via two sequential steps: the adsorption of a temperature- and pH-sensitive microgel synthesized by precipitation copolymerization of N-isopropylacrylamide (NIPAM) and 3-(N,N-dimethylamino)propylmethacrylamide (DMAPMA) (poly(NIPAM-co-DMAPMA) at the pH-condition corresponding to its noncharged state (first step of adsorption), followed by the enzyme, tyrosinase, adsorption at the pH-condition when the microgel and the enzyme are oppositely charged (second step of adsorption). The stimuli-sensitive properties of poly(NIPAM-co-DMAPMA) microgel were characterized by potentiometric titration and dynamic light scattering (DLS) in solution as well as by atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) at solid interface. Enhanced deposition of poly(NIPAM-co-DMAPMA) microgel particles was shown at elevated temperatures exceeding the volume phase transition temperature (VPTT). The subsequent electrostatic interaction of the poly(NIPAM-co-DMAPMA) microgel matrix with tyrosinase was examined at different adsorption regimes. A considerable increase in the amount of the adsorbed enzyme was detected when the microgel film is first brought into a collapsed state but then was allowed to interact with the enzyme at T < VPTT. Spongelike approach to enzyme adsorption was applied for modification of screen-printed graphite electrodes by poly(NIPAM-co-DMAPMA)/tyrosinase films and the resultant biosensors for phenol were tested amperometrically. By temperature-induced stimulating both (i) poly(NIPAM-co-DMAPMA) microgel adsorption at T > VPTT and (ii) following spongelike tyrosinase loading at T < VPTT, we can achieve more than 3.5-fold increase in biosensor sensitivity for phenol assay. Thus, a very simple, novel, and fast strategy for physical entrapment of biomolecules by the polymeric matrix was proposed and tested. Being based on this unique stimuli-sensitive behavior of the microgel, this stimulated spongelike adsorption provides polymer films comprising concentrated biomaterial.

In Situ SERS Sensing by a Laser-Induced Aggregation of Silver Nanoparticles Templated on a Thermoresponsive Polymer.

A stimuli-responsive (pH- and thermoresponsive) micelle-forming diblock copolymer, poly(1,2-butadiene)290-block-poly(N,N-dimethylaminoethyl methacrylate)240 (PB-b-PDMAEMA), was used as a polymer template for the in situ synthesis of silver nanoparticles (AgNPs) through Ag+ complexation with PDMAEMA blocks, followed by the reduction of the bound Ag+ with sodium borohydride. A successful synthesis of the AgNPs on a PB-b-PDMAEMA micellar template was confirmed by means of UV-Vis spectroscopy and transmission electron microscopy, wherein the shape and size of the AgNPs were determined. A phase transition of the polymer matrix in the AgNPs/PB-b-PDMAEMA metallopolymer hybrids, which results from a collapse and aggregation of PDMAEMA blocks, was manifested by changes in the transmittance of their aqueous solutions as a function of temperature. A SERS reporting probe, 4-mercaptophenylboronic acid (4-MPBA), was used to demonstrate a laser-induced enhancement of the SERS signal observed under constant laser irradiation. The local heating of the AgNPs/PB-b-PDMAEMA sample in the laser spot is thought to be responsible for the triggered SERS effect, which is caused by the approaching of AgNPs and the generation of "hot spots" under a thermo-induced collapse and the aggregation of the PDMAEMA blocks of the polymer matrix. The triggered SERS effect depends on the time of a laser exposure and on the concentration of 4-MPBA. Possible mechanisms of the laser-induced heating for the AgNPs/PB-b-PDMAEMA metallopolymer hybrids are discussed.

Simultaneous express immunoassay of multiple cardiac biomarkers with an automatic platform in human plasma.

C-reactive protein, cystatin C, myoglobin, and D-dimer represent the inflammatory or thromboembolic status of the patient and play important roles in early diagnostics of acute myocardial infarction. Each protein can indicate some health problems, but their simultaneous detection can be crucial for differential diagnostics. The express analysis of these proteins in a small drop of plasma was developed using magnetic beads. The suggested method is based on immunomagnetic extraction of the target analyte from plasma samples and its simultaneous labelling by fluorescent dye. Reaction time was optimized for quantification of cardiac biomarkers in the spike solutions and human plasma samples. In this paper, we developed a one-protein detection technique for each cardiac biomarker and united it to a four-protein facility using an automatic platform. The proposed technique requires only 17 µL of the human plasma and takes 14 min for four-protein measuring. The suggested technique covers concentration difference by more than two orders of magnitude and demonstrates analytical applicability by measurements of human plasma samples of 16 volunteers.

Cyclic on-chip bacteria separation and preconcentration.

Nanoparticles and biological molecules high throughput robust separation is of significant interest in many healthcare and nanoscience industrial applications. In this work, we report an on-chip automatic efficient separation and preconcentration method of dissimilar sized particles within a microfluidic platform using integrated membrane valves controlled microfiltration. Micro-sized E. coli bacteria are sorted from nanoparticles and preconcentrated on a microfluidic chip with six integrated pneumatic valves (sub-100 nL dead volume) using hydrophilic PVDF filter with 0.45 µm pore diameter. The proposed on-chip automatic sorting sequence includes a sample filtration, dead volume washout and retentate backflush in reverse flow. We showed that pulse backflush mode and volume control can dramatically increase microparticles sorting and preconcentration efficiency. We demonstrate that at the optimal pulse backflush regime a separation efficiency of E. coli cells up to 81.33% at a separation throughput of 120.45 µL/min can be achieved. A trimmed mode when the backflush volume is twice smaller than the initial sample results in a preconcentration efficiency of E. coli cells up to 121.96% at a throughput of 80.93 µL/min. Finally, we propose a cyclic on-chip preconcentration method which demonstrates E. coli cells preconcentration efficiency of 536% at a throughput of 1.98 µL/min and 294% preconcentration efficiency at a 10.9 µL/min throughput.

Multiscale flaked silver SERS-substrate for glycated human albumin biosensing.

Original multiscale flaked silver SERS-substrate (MFSS substrate) was applied for glycated albumin (GA) biosensing. The substrate is composed from silver flakes that have three orders of magnitude size dispersion: from 50 nm to 2 µm. The multiscale silver structure refracts the incident light and various surface plasmons are excited. Some of the internal plasmons are localized and give rise of the large local electric field. It was demonstrated that Raman scattering signal strongly depends: a) on "hot spots" formation at the edges and points of contact of silver plates, and b) on the angle of incidence. As a result the silver structure operates as an effective SERS substrate. To achieve the selectivity to glycated part, the surface of SERS-substrate was modified with 4-mercaptophenylboronic acid (4-mPBA). Various saccharides (Fru, Glc, Suc, Dex) were taken as model compounds for the glycated proteins determination. The saccharides contain cis-diol groups that form five- or six-membered ethers with boronic acid. Spectrum of SERS-substrate changes after sugar/glycated albumin treatment. Main differences in the SERS-spectra of sugar/glycated albumin treated SERS-substrate and control are referred to phenylboronic acid vibrations (999, 1021, 1072 and 1589 cm-1). Principal component analysis (PCA) and Partial Least Squares Regression (PLS-R) were used to discriminate spectra and to construct calibration curve, as well as to measure GA values in real samples of human plasma. Multiscale flaked silver SERS-substrate modified with 4-mPBA allows quantitative one-step biosensing of glycated albumin in 15 µl of human plasma.

Surface Functionalization by Stimuli-Sensitive Microgels for Effective Enzyme Uptake and Rational Design of Biosensor Setups.

We highlight microgel/enzyme thin films that were deposited onto solid interfaces via two sequential steps, the adsorption of temperature- and pH-sensitive microgels, followed by their complexation with the enzyme choline oxidase, ChO. Two kinds of functional (ionic) microgels were compared in this work in regard to their adsorptive behavior and interaction with ChO, that is, poly(N-isopropylacrylamide-co-N-(3-aminopropyl)methacrylamide), P(NIPAM-co-APMA), bearing primary amino groups, and poly(N-isopropylacrylamide-co-N-[3-(dimethylamino) propyl]methacrylamide), P(NIPAM-co-DMAPMA), bearing tertiary amino groups. The stimuli-sensitive properties of the microgels in the solution were characterized by potentiometric titration, dynamic light scattering (DLS), and laser microelectrophoresis. The peculiarities of the adsorptive behavior of both the microgels and the specific character of their interaction with ChO were revealed by a combination of surface characterization techniques. The surface charge was characterized by electrokinetic analysis (EKA) for the initial graphite surface and the same one after the subsequent deposition of the microgels and the enzyme under different adsorption regimes. The masses of wet microgel and microgel/enzyme films were determined by quartz crystal microbalance with dissipation monitoring (QCM-D) upon the subsequent deposition of the components under the same adsorption conditions, on a surface of gold-coated quartz crystals. Finally, the enzymatic responses of the microgel/enzyme films deposited on graphite electrodes to choline were tested amperometrically. The presence of functional primary amino groups in the P(NIPAM-co-APMA) microgel enables a covalent enzyme-to-microgel coupling via glutar aldehyde cross-linking, thereby resulting in a considerable improvement of the biosensor operational stability.

Biosensor assay of neuropathy target esterase in whole blood as a new approach to OPIDN risk assessment: review of progress.

Organophosphates (OPs) that inhibit neuropathy target esterase (NTE) with subsequent ageing can produce OP-induced delayed neuropathy (OPIDN). NTE inhibition in lymphocytes can be used as a biomarker of exposure to neuropathic OPs. An electrochemical method was developed to assay NTE in whole blood. The high sensitivity of the tyrosinase carbon-paste biosensors for the phenol produced by hydrolysis of the substrate, phenyl valerate, allowed NTE activity to be measured in diluted samples of whole blood, which cannot be done using the standard colorimetric assay. The biosensor was used to establish correlations of NTE inhibitions in blood with that in lymphocytes and brain after dosing hens with a neuropathic OP. The results of further studies demonstrated that whole blood NTE is a reliable biomarker of neuropathic OPs for up to 96 hours after exposure. These validation results suggest that the biosensor NTE assay for whole blood could be developed to measure human exposure to neuropathic OPs as a predictor of OPIDN. The small blood volume required (100 microL), simplicity of sample preparation and rapid analysis times indicate that the biosensor should be useful in biomonitoring and epidemiological studies. The present paper is an overview of our previous and ongoing work in this area.

Biosensor detection of neuropathy target esterase in whole blood as a biomarker of exposure to neuropathic organophosphorus compounds.

Neuropathy target esterase (NTE) is the target protein for neuropathic organophosphorus (OP) compounds that produce OP compound-induced delayed neurotoxicity (OPIDN). Inhibition/aging of brain NTE within hours of exposure predicts the potential for development of OPIDN in susceptible animal models. Lymphocyte NTE has also found limited use as a biomarker of human exposure to neuropathic OP compounds. Recently, a highly sensitive biosensor was developed for NTE activity using a tyrosinase carbon-paste electrode for amperometric detection of phenol produced by hydrolysis of the substrate, phenyl valerate. The I50 (20 min at 37 degrees C) for N,N'-di-2-propylphosphorodiamidofluoridate (mipafox) against hen lymphocyte NTE was 6.94 +/- 0.28 microM amperometrically and 6.02 +/- 0.71 microM colorimetrically. For O,O-di1-propyl O-2,2-dichlorvinyl phosphate (PrDChVP), the I50 against hen brain NTE was 39 +/- 8 nM amperometrically and 42 +/- 2 nM colorimetrically. The biosensor enables NTE to be assayed in whole blood, whereas this cannot be done with the usual colorimetric method. Amperometrically, I50 values for PrDChVP against hen and human blood NTE were 66 +/- 3 and 70 +/- 14 nM, respectively. To study the possibility of using blood NTE inhibition as a biochemical marker of neuropathic OP compound exposure, NTE activities in brain and lymphocytes as well in brain and blood were measured 24 h after dosing hens with PrDChVP. Brain, lymphocyte, and blood NTE were inhibited in a dose-responsive manner, and NTE inhibition was highly correlated between brain and lymphocyte (r = .994) and between brain and blood (r = .997). The results suggest that the biosensor NTE assay for whole blood could serve as a biomarker of exposure to neuropathic OP compounds as well as a predictor of OPIDN and an adjunct to its early diagnosis.

Sequential pH-dependent adsorption of ionic amphiphilic diblock copolymer micelles and choline oxidase onto conductive substrates: toward the design of biosensors.

This work examines the fabrication regime and the properties of polymer-enzyme thin-films adsorbed onto conductive substrates (graphite or gold). The films are formed via two-steps, sequential adsorption of poly(n-butylmethacrylate)-block-poly(N,N-dimethylaminoethyl methacrylate) (PnBMA-b-PDMAEMA) diblock copolymer micelles (1st step of adsorption), followed by the enzyme choline oxidase (ChO) (2nd step of adsorption). The solution properties of both adsorbed components are studied and the pH-dependent step-by-step fabrication of polymer-enzyme biosensor coatings reveals rather drastic differences in their enzymatic activities in dependence on the pH of both adsorption steps. The resulting hybrid thin-films represent highly active biosensors for choline with a low detection limit of 30 nM and a good linearity in a range between 30 nM and 100 µM. The sensitivity is found to be 175 µA mM(-1) cm(-2) and the operational stability of the polymer-enzyme thin-films can be additionally improved via enzyme-to-enzyme crosslinking with glutaraldehyde.

Prussian blue based nanoelectrode arrays for H(2)O(2) detection.

We propose to form nanoelectrode arrays by deposition of the electrocatalyst through lyotropic liquid crystalline templates onto inert electrode support. Whereas Prussian Blue is known to be a superior electrocatalyst in hydrogen peroxide reduction, carbon materials used as electrode support demonstrate only a minor activity. We report on the possibility for nanostructuring of Prussian Blue by its electrochemical deposition through lyotropic liquid crystalline templates, which is noticed from atomic force microscopy images of the resulting surfaces. The resulting Prussian Blue based nanoelectrode arrays in flow injection analysis mode demonstrate a sub-part-per-billion detection limit (1 x 10(-)(8) M) and a linear calibration range starting exactly from the detection limit and extending over 6 orders of magnitude of H(2)O(2) concentrations (1 x 10(-)(8) to 1 x 10(-)(2) M), which are the most advantageous analytical performances in hydrogen peroxide electroanalysis.