Is infrared spectroscopy combined with multivariate analysis a promising tool for heparin authentication?

The investigation of the possibility to determine various characteristics of powder heparin (n = 115) was carried out with infrared spectroscopy. The evaluation of heparin samples included several parameters such as purity grade, distributing company, animal source as well as heparin species (i.e. Na-heparin, Ca-heparin, and heparinoids). Multivariate analysis using principal component analysis (PCA), soft independent modelling of class analogy (SIMCA), and partial least squares - discriminant analysis (PLS-DA) were applied for the modelling of spectral data. Different pre-processing methods were applied to IR spectral data; multiplicative scatter correction (MSC) was chosen as the most relevant. Obtained results were confirmed by nuclear magnetic resonance (NMR) spectroscopy. Good predictive ability of this approach demonstrates the potential of IR spectroscopy and chemometrics for screening of heparin quality. This approach, however, is designed as a screening tool and is not considered as a replacement for either of the methods required by USP and FDA.

Quality Control of Heparin Injections: Comparison of Four Established Methods.

Heparin is an anticoagulant medication that is usually injected subcutaneously. The quality of a set of commercial heparin injections from different producers was examined by NMR, IR, UV-Vis spectroscopies and potentiometric multisensor system. The type of raw material regarding heparin animal origin and producer, heparin molecular weight and activity values were derived based on the non-targeted analysis of 1H NMR fingerprints. DOSY NMR spectroscopy was additionally used to study homogeneity and additives profile. UV-Vis and IR, being cheaper than NMR, combined with multivariate statistics were successfully applied to study excipients composition as well as semi-estimation of activity values. Potentiometric multisensor measurements were found to be an important additional source of information about inorganic composition of finished heparin formulations. All investigated instrumental techniques are useful for finished heparin injections and should be selected according to availability as well as the information and confidence required for a specific sample.

Molecularly imprinted polyaniline for detection of horseradish peroxidase.

A new facile and fast approach to the synthesis of polyaniline (PANi) molecularly imprinted polymers (MIPs) based on aniline oxidative chemical polymerization was proposed for protein recognition. For the first time, a surface imprinting strategy was implemented for the synthesis of PANi MIPs on the inner surface of soft glass polycapillaries (PC) with a large (2237) number of individual microcapillaries. Two different PANi layers-(i) PANi film and (ii) protein imprinted PANi nanowires-were synthesized sequentially. Uniform and highly stable PANi film was synthesized by oxidative polymerization at pH< 1. The synthesis of PANi MIPs on the PANi film pre-coated surface improved the reproducibility of PANi MIP formation. PANi MIP nanowires were synthesized at "mild" conditions (pH > 4.5) to preserve the protein template activity. The binding of horseradish peroxidase (HRP) molecules on the PANi MIP selective sites was confirmed by photometry (TMB chromogenic reaction), SEM images, and FTIR spectroscopy. The developed PANi MIPs enable HRP determination with a limit of detection (LOD) as low as 1.00 and 0.07 ng mL-1 on the glass slips and PC, respectively. The PANi MIPs are characterized by high stability; they are reversible and selective to HRP. The proposed approach allows PANi MIPs to be obtained for proteins on different supports and to create new materials for separation and sensing. Graphical abstract.

Optical sensors for determination of biogenic amines in food.

This review presents the state-of-the-art of optical sensors for determination of biogenic amines (BAs) in food by publications covering about the last 10 years. Interest in the development of rapid and preferably on-site methods for quantification of BAs is based on their important role in implementation and regulation of various physiological processes. At the same time, BAs can develop in different kinds of food by fermentation processes or microbial activity or arise due to contamination, which induces toxicological risks and food poisoning and causes serious health issues. Therefore, various optical chemosensor systems have been devised that are easy to assemble and fast responding and low-cost analytical tools. If amenable to on-site analysis, they are an attractive alternative to existing instrumental analytical methods used for BA determination in food. Hence, also portable sensor systems or dipstick sensors are described based on various probes that typically enable signal readouts such as photometry, reflectometry, luminescence, surface-enhanced Raman spectroscopy, or ellipsometry. The quantification of BAs in real food samples and the design of the sensors are highlighted and the analytical figures of merit are compared. Future instrumental trends for BA sensing point to the use of cell phone-based fully automated optical evaluation and devices that could even comprise microfluidic micro total analysis systems.

Soft glass multi-channel capillaries as a platform for bioimprinting.

Multi-channel capillaries (MC) formed from thousands individual microcapillaries with diameters ranging 10-100 µm are of a great interest for their use as platforms for molecular imprinting due to their relatively large surface area, high mechanical stability and possibility of facile integration in sensor systems. The manuscript proposes a new format of immunoassay based on imprinted protein immobilized on a MC inner surface modified with poly-l-lysine. The combination of the environmentally friendly, easy-to-produce and cheap recognition element with the carrier allowing to increase the assay sensitivity makes the described technique a perspective alternative for the existing screening tests. Two bioimprinting approaches were described. The imprinted protein (ovalbumin, OVA) primarily prepared separately and later immobilized on a MC structure was compared to the imprinted OVA directly prepared on the MC surface. Detection of a food contaminant zearalenone was chosen as a proof-of-concept. In a case of the immobilization of the primarily prepared imprinted OVA the reached limit of detection (LOD) was 0.8 ng/mL, and for the in-situ imprinted OVA the LOD was 0.12 ng/mL. The sensitivity of the developed bioimprinted assay was comparable to the commercially available ELISA kits for ZEN detection. The OVA in-situ imprinted on the MC surface was tested for the detection of ZEN in artificially spiked wheat samples. The high recovery values (88-112%) and good repeatability (RSD of 8.5-9.6%) were demonstrated allowing to conclude that the IPs-based MC-ELISA is a promising tool for analysis of the mycotoxin in complex matrices.

Simultaneous determination of proteins in microstructured optical fibers supported by chemometric tools.

A new perspective on the relevant problem-creating simple, rapid, and efficient protein sensors based on microstructured optical fibers using a simple homogeneous analysis format-was proposed. Commercially available long-period grating hollow core microstructured optical fibers (LPG HCMOF) were used to determine bovine serum albumin (BSA) and albumin from chicken eggs (OVA) in binary mixtures as well as immunoglobulin G (IgG) in the presence of BSA and OVA. LPG HCMOF transmission spectra allowed the detection of both BSA and OVA up to 10 mg/mL with LOD as low as 0.1 and 0.8 µg/mL, respectively. Partial least squares regression (PLS) was utilized for modeling of LPG HCMOF spectral data and quantitative analysis of BSA, OVA, total protein, and IgG in binary and ternary mixtures. Rather high coefficients of determination (R2) and low root mean square error for the calibration (RMSEC) (15%) and prediction (RMSEP) (20%) were obtained for all PLS models. The proposed approach was tested in the analysis of BSA in spiked horse blood hemolyzed (HBH). The results demonstrated the functionality of the proposed approach and offered the opportunity for the creation of a wide range of sensors for protein determination in complex mixtures. Graphical abstract.

Microstructured Waveguides with Polyelectrolyte-Stabilized Gold Nanostars for SERS Sensing of Dissolved Analytes.

A sensor based on microstructured waveguides (MWGs) with a hollow core inner surface covered with polyelectrolyte-layer-stabilized gold nanostars was developed for the SERS sensing of dissolved analytes. A polyelectrolyte-layer coating over the inner surface of glass cladding served as a spacer, reducing nonlinear optical effects in the glass near plasmonic hotspots of nanoparticles, as a stabilizing agent for thermodynamically unstable gold nanostars and as an optical coating for the fine-tuning of MWG bandgaps. This approach can be used to construct different kinds of SERS sensors for dissolved analytes, providing conservation, the prevention of coagulation, and the drying of a liquid sample for the time required to record the signal.

Microstructured optical fiber-based luminescent biosensing: Is there any light at the end of the tunnel? - A review.

This review covers the current state of the art of luminescent biosensors based on various types of microstructured optical fiber. The unique optical and structural properties of this type of optical fiber make them one of the most promising integrated platforms for bioassays. The individual sections of this review are devoted to a) classification of microstructured optical fibers, b) microstructured optical fiber materials, c) aspects of biosensing based on the biomolecules incorporated into the microstructured optical fibers, and d) development of models for prediction of the efficiency of luminescent signal processing. The authors' views on current trends and limitations of microstructured optical fibers for biosensing as well as the most promising areas and technologies for application in analytical practice are presented.

An immunochemical test for rapid screening of zearalenone and T-2 toxin.

An immunochemically based test for non-instrumental simultaneous detection of zearalenone (ZEA) and T-2 toxin (T2) in feed was developed. The method combines clean-up of sample extract, pre-concentration of analytes by immunoextraction and immunodetection through the enzymatic reaction of horseradish peroxidase (HRP). The test is housed inside a standard 1-mL solid-phase extraction column and consists of three layers: two test layers (one for ZEA and another for T2) with immobilised specific antibodies and one control layer with bound anti-HRP antibodies. Feed extract was passed through an additional column with clean-up layer, which was disconnected after extract application. Total assay time was about 15 min for six samples and detection time was 4 min after chromogenic substrate application. Under optimised conditions a cut-off level for ZEA and T2 of 100 microg/kg was established. Different feed types were analysed for ZEA and T2 contamination by the proposed method and results were confirmed by LC-MS/MS.

Application of a new anti-zearalenone monoclonal antibody in different immunoassay formats.

Monoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based on the competitive direct enzyme immunoassay principle were developed: an enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay. In ELISA, the 50% inhibitory concentration (IC50) was 0.8 ng/mL, and the limit of detection for ZEA standard solutions was 0.1 ng/mL. The antibodies showed a high ZEA (100%) and alpha-zearalenol (alpha-ZOL) (69%) recognition, while cross-reactivities with alpha-zearalanol, zearalanone, beta-zearalenol and beta- zearalanol were 42%, 22%, <1% and <1%, respectively. For standard solutions, a cut-off level at 10 ng/mL could be established for the gel- and membrane-based enzyme immunoassays. Assay time of both non-instrumental tests was 25 min for 10 samples. By including a simple sample extraction procedure, the methods were applied to wheat with IC50s in ELISA of 80 and 120 microg/kg (dilution up to 5% and 15% (v/v) of wheat matrix, respectively). The cut-off level of the gel- and membrane-based immunoassays was established at 100 microg/kg. Potentials and limitations of the developed methods were compared. The possible application for multi-mycotoxin analysis of the ELISA method based on a single monoclonal antibody was investigated. Therefore, principal component analysis and partial least squares regression data modelling were used to separate the immunoassay responses of two cross-reactants (ZEA and alpha-ZOL).

Rapid method for qualitative detection of in environmental samples.

A gel-based immunoassay that can be used for the detection of 2,4,6-trinitrotoluene (TNT) in water samples was developed. Four polyclonal antibodies were generated in chickens using TNT derivatives. The assay was based on the immunoaffinity preconcentration and immuno-enzyme analysis of TNT in the gel. The results of the assay, assessed by color development, were evaluated visually and also by using a flatbed scanner and subsequent digital processing of the scanned gel. The most sensitive color mode, parameter S (saturation, HSB mode), was used for the immunoassay optimization and evaluation of the results. The immunoassays with the best parameters were optimized and characterized. A cut-off level of 5 µg TNT L-1 was reached for water samples. It was shown that tap and environmental water samples could be analyzed directly, without sample preparation and dilution. The developed test is acceptable for use in an on-site field test to provide rapid (about 15 min for six samples), qualitative and reliable results for making environmental decisions such as identifying "hot spots", monitoring of military and terrorist activities, and selecting of site samples for laboratory analysis.