Simultaneous determination of L- and D-methotrexate using a sequential injection analysis/amperometric biosensors system

A sequential injection analysis (SIA) is proposed for the simultaneous determination of L- and D-methotrexate (Mtx) using amperometric biosensors as detectors. A SIA system is proposed due to the highest precision and accuracy and lower consumption of sample and buffer. The amperometric biosensors used as detectors in SIA system were based on L-amino acid oxidase (L-AAOD) or/and L-glutamate oxidase (L-Glox) and horseradish peroxidase (HRP) for the assay of L-Mtx and D-amino acid oxidase (D-AAOD) and HRP for the assay of D-Mtx were selected. The linear concentration ranges are of pmol/l to nmol/l magnitude order, with very low limits of detection. The SIA/biosensors system can be used reliably on-line in synthesis process control, for the simultaneous assay of L- and D-Mtx with a frequency of 34 samples per hour.     

Simultaneous detection of creatine and creatinine using a sequential injection analysis/biosensor system

Carbon paste based biosensors for the determination of creatine and creatinine have been integrated into a sequential injection system. Applying the multi-enzyme sequence of creatininase (CA), and/or creatinase (CI) and sarcosine oxidase (SO), hydrogen peroxide has been detected amperometrically. The linear concentration ranges are of pmol/L to nmol/L magnitude, with very low limits of detection. The proposed SIA system can be utilized reliably for the on-line simultaneous detection of creatine and creatinine in pharmaceutical products, as well as in serum samples, with a rate of 34 samples per hour and RSD values better than 0.16% (n=10).     

Simultaneous Detection of Creatine and Creatinine using a Sequential Injection Analysis/Biosensor System

Abstract

Carbon paste based biosensors for the determination of creatine and creatinine have been integrated into a sequential injection system. Applying the multi‐enzyme sequence of creatininase (CA), and/or creatinase (CI) and sarcosine oxidase (SO), hydrogen peroxide has been detected amperometrically. The linear concentration ranges are of pmol/L to nmol/L magnitude, with very low limits of detection. The proposed SIA system can be utilized reliably for the on‐line simultaneous detection of creatine and creatinine in pharmaceutical products, as well as in serum samples, with a rate of 34 samples per hour and RSD values better than 0.16% (n=10).     

Determination of codeine and metabolites in plasma and urine using ion-pair high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method for the simultaneous determination of codeine and seven metabolites is described. The samples are purified by reversed-phase solid-phase extraction. Codeine, norcodeine, codeine-6-glucuronide, norcodeine-6-glucuronide and morphine-3-glucoronide are measured with UV detection. Detection limits are 3 nmol/l (morphine-3-glucuronide) to 20 nmol/l (codeine). Morphine, normorphine and morphine-6-glucuronide are measured with electrochemical detection. Detection limits are 0.4 nmol/l (morphine-6-glucuronide) to 1.0 nmol/l (normorphine). Correlation coefficients better than 0.998 are normally obtained for all compounds. The method was applied to the determination of the kinetics of codeine and its metabolites in plasma and urine samples from healthy volunteers.     

Amperometric ATP biosensor based on polymer entrapped enzymes

A dual enzyme electrode for the detection of adenosine-5'-triphosphate (ATP) at physiologically relevant pH levels was developed by co-immobilization of the enzymes glucose oxidase (GOD) and hexokinase (HEX) using pH-shift induced deposition of enzyme containing polymer films. Application of a simple electrochemical procedure for the co-immobilization of the enzymes at electrode surfaces exhibits a major improvement of sensitivity, response time, reproducibility, and ease of fabrication of ATP biosensors. Competition between glucose oxidase and hexokinase for the substrate glucose involving ATP as a co-substrate allows the determination of ATP concentrations. Notable control on the immobilization process enables fabrication of micro biosensors with a diameter of 25 microm. The presented concept provides the technological basis for a new generation of fast responding, sensitive, and robust biosensors for the detection of ATP at physiological pH values with a detection limit of 10 nmol l(-1).     

Improved multianalyte detection of organophosphates and carbamates with disposable multielectrode biosensors using recombinant mutants of Drosophila acetylcholinesterase and artificial neural networks

Engineered variants of Drosophila melanogaster acetylcholinesterase (AChE) were used as biological receptors of AChE-multisensors for the simultaneous detection and discrimination of binary mixtures of cholinesterase-inhibiting insecticides. The system was based on a combination of amperometric multielectrode biosensors with chemometric data analysis of sensor outputs using artificial neural networks (ANN). The multisensors were fully manufactured by screen-printing, including enzyme immobilisation. Two types of multisensors were produced that consisted of four AChE variants each. The AChE mutants were selected in order to obtain high resolution, enhanced sensitivity and minimal assay time. This task was successfully achieved using multisensor I equipped with wild-type Drosophila AChE and mutants Y408F, F368L, and F368H. Each of the AChE variants was selected on the basis of displaying an individual sensitivity pattern towards the target analytes. For multisensor II, the inclusion of F368W, which had an extremely diminished paraoxon sensitivity, increased the sensor's capacity even further. Multisensors I and II were both used for inhibition analysis of binary paraoxon and carbofuran mixtures in a concentration range 0-5 microg/l, followed by data analysis using feed-forward ANN. The two analytes were determined with prediction errors of 0.4 microg/l for paraoxon and 0.5 microg/l for carbofuran. A complete biosensor assay and subsequent ANN evaluation was completed within 40 min. In addition, multisensor II was also investigated for analyte discrimination in real water samples. Finally, the properties of the multisensors were confirmed by simultaneous detection of binary organophosphate mixtures. Malaoxon and paraoxon in composite solutions of 0-5 microg/l were discriminated with predication errors of 0.9 and 1.6 microg/l, respectively.     

Simultaneous stereoselective analysis of venlafaxine and O-desmethylvenlafaxine enantiomers in human plasma by HPLC-ESI/MS using a vancomycin chiral column

A high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI/MS) method for simultaneous stereoselective analysis of venlafaxine (VEN) and its major metabolite O-desmethylvenlafaxine (ODV) enantiomers in human plasma has been developed and validated. Chiral chromatography is performed on the CHRIOBIOTIC V (5 microm, 250 mm x 4.6 mm) column with mobile phase constituted of 30 mmol/l ammonium acetate-methanol (15:85, pH 6.0) at a flow rate of 1.0 ml/min and a postcolumn splitting ratio of 3:1. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and detected using the selected ion recording (SIR) mode. Calibration curves obtained from spiked plasma were linear in the range of 5.0-400 ng/ml for S-(+)-VEN and R-(-)-VEN, 4.0-280 ng/ml for S-(+)-ODV and R-(-)-ODV, respectively, with linear correlation coefficient all above 0.999. The average extraction recoveries for all the four analytes were above 76%. The methodology recoveries were higher than 92%. The limit of detection were 1.0 ng/ml for S-(+)-VEN and R-(-)-VEN, 1.5 ng/ml for S-(+)-ODV and R-(-)-ODV, respectively. The intra- and inter-day variation coefficients were less than 9%.     

Optimisation and validation of a sensitive high-performance liquid chromatography assay for routine measurement of pyridoxal 5-phosphate in human plasma and red cells using pre-column semicarbazide derivatisation

There are few studies in which direct measurement of vitamin B6 status in both plasma and red cells has been assessed. The aims of the present study were to evaluate the use of a simple, robust HPLC method of direct pyridoxal 5'-phosphate (PLP) measurement in plasma and red cells and to assess its use in establishing reference ranges in a healthy population. A reverse phase HPLC method with pre-column derivatisation using semicarbazide for the simultaneous measurement of PLP, its degradation product, 4-pyridoxic acid (PA) and pyridoxal (PL) in plasma and red cells was developed. Pre-column derivatisation, reverse phase chromatography and detection procedures were optimised. The recovery, precision, linearity and sensitivity of the assay for plasma and red cell PLP, PA and PL was established. The recovery of PLP was greater than 95% for both plasma and red cell samples. The Intra and Inter batch imprecision for PLP was less than 6% and 7%, respectively. The method for PLP was linear up to at least 1000 nmol/l and the detection limit was 2.1 nmol/l (limit of quantification; 5.8 nmol/l). Accuracy of PLP measurements in plasma were acceptable, showing a mean bias of 4.5% from the mean value of laboratories (N=34) participating in an external quality assurance scheme. Geometric mean (95% reference intervals) for plasma and red cell PLP in the healthy subjects (N=126) were 56 (21-138) nmol/l and 410 (250-680) pmol/g Hb, respectively. There was a strong positive correlation (r(2)=0.81) between plasma and red cell PLP levels in the reference population. The HPLC method described was found to be suitable for the routine measurement of PLP in both plasma and red cells.     

Biosensors for the determination of ortho-acetyl-L-carnitine. Their utilization as detectors in a sequential injection analysis system

In order to determine ortho-acetyl-L-carnitine, two biosensors were proposed. The biosensors were designed using physical immobilization of L-amino acid oxidase (L-AAOD) and horseradish peroxidase (HRP). Electrode characteristics were obtained and compared for the two carbon paste (graphite powder and paraffin oil) biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol/L to nmol/L, magnitude order with low limits of detection. Due to their reliability, the biosensors were used as detectors in a sequential injection analysis system, and gave reliable results for on-line assay of ortho-acetyl-L-carnitine in synthesis process control with a frequency of 75 samples per hour.     

The Sonogel-Carbon materials as basis for development of enzyme biosensors for phenols and polyphenols monitoring: a detailed comparative study of three immobilization matrixes

Three amperometric biosensors based on immobilization of tyrosinase on a new Sonogel-Carbon electrode for detection of phenols and polyphenols are described. The electrode was prepared using high energy ultrasounds (HEU) directly applied to the precursors. The first biosensor was obtained by simple adsorption of the enzyme on the Sonogel-Carbon electrode. The second and the third ones, presenting sandwich configurations, were initially prepared by adsorption of the enzyme and then modification by mean of polymeric membrane such as polyethylene glycol for the second one, and the ion-exchanger Nafion in the case of the third biosensor. The optimal enzyme loading and polymer concentration, in the second layer, were found to be 285 U and 0.5%, respectively. All biosensors showed optimal activity at the following conditions: pH 7, -200 mV, and 0.02 mol l(-1) phosphate buffer. The response of the biosensors toward five simple phenols derivatives and two polyphenols were investigated. It was found that the three developed tyrosinase Sonogel-Carbon based biosensors are in satisfactory competitiveness for phenolic compounds determination with other tyrosinase based biosensors reported in the literature. The detection limit, sensitivity, and the apparent Michaelis-Menten constant K(m)(app) for the Nafion modified biosensor were, respectively, 0.064, 0.096, and 0.03 micromol, 82.5, 63.4, and 194 nA micromol(-1)l(-1), and 67.1, 54.6, and 12.1 micromol l(-1) for catechol, phenol, and 4-chloro-3-methylphenol. Hill coefficient values (around 1 for all cases), demonstrated that the immobilization method does not affect the nature of the enzyme and confirms the biocompatibility of the Sonogel-Carbon with the bioprobe. An exploratory application to real samples such as beers, river waters and tannery wastewaters showed the ability of the developed Nafion/tyrosinase/Sonogel-Carbon biosensor to retain its stable and reproducible response.     

Simultaneous liquid chromatographic assessment of thiamine,thiamine monophosphate and thiamine diphosphate in human erythrocytes: a study on alcoholics

An isocratic HPLC procedure for the assessment of thiamine (T), thiamine monophosphate (TMP) and thiamine diphosphate (TDP) in human erythrocytes is described. Several aspects of the procedure make it suitable for both clinical and research purposes: limits of detection and quantification of 1 and 2.5 nmol/l, respectively, recovery of 102% on average (range 93-112%), intra- and inter-day precisions within 5 and 9%, respectively, total elution time 15 min. This analytical methodology was applied to a case-control study on erythrocyte samples from 103 healthy subjects and 36 alcohol-dependent patients at risk of thiamine deficiency. Mean control values obtained were: T=89.6+/-22.7 nmol/l, TMP=4.4+/-6.6 nmol/l and TDP=222.23+/-56.3 nmol/l. T and TDP mean values of alcoholics were significantly lower than those of control cases: T=69.4+/-35.9 nmol/l (P<0.001) and TDP=127.4+/-62.5 nmol/l (P<10(-5)). The diagnostic role of TDP was evaluated and a significant role for thiamine was established in the study of alcohol related problems.     

Biosensors for the Determination of Ortho-acetyl-L-carnitine. Their Utilization as Detectors in a Sequential Injection Analysis System

Abstract

In order to determine ortho-acetyl-L-carnitine, two biosensors were proposed. The biosensors were designed using physical immobilization of L-amino acid oxidase (L-AAOD) and horseradish peroxidase (HRP). Electrode characteristics were obtained and compared for the two carbon paste (graphite powder and paraffin oil) biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol/L to nmol/L, magnitude order with low limits of detection. Due to their reliability, the biosensors were used as detectors in a sequential injection analysis system, and gave reliable results for on-line assay of ortho-acetyl-L-carnitine in synthesis process control with a frequency of 75 samples per hour.     

Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists

A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a Solid-Phase Extraction column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post exercise versus pre exercise condition in cyclists (F: 136+/-93 nmol/l versus 67+/-50 nmol/l (p<0.001); E: 797+/-400 nmol/l versus 408+/-252 nmol/l (p<0.001)). The F/E ratio was 0.18+/-0.11 versus 0.16+/-0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11+/-0.07) and cyclist individuals at rest (p<0.05).     

Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography

A new and reliable HPLC method for the quantitation of daidzein, equol, and genistein in human breast tissue has been developed. The method was applied to biopsies from women undergoing breast reductions, who, prior to surgery, had ingested either a soy isoflavone preparation or a placebo tablet. The results were compared with data collected for urine and serum of the same subjects using standard methods. The limits of detection in the breast tissue homogenate were 24.7 nmol/l for daidzein, 148.0 nmol/l for equol, and 28.4 nmol/l for genistein (S/N of 3). The chromatographic limits of quantitation were 62.5 nmol/l for daidzein and genistein, and 125.0 nmol/l for equol, for which the accuracies were 86.0%, 83.6%, and 81.8%, respectively. The coefficients of variation of these measurements were all below 20% (11.1% for daidzein, 16.4% for genistein, and 13.2% for equol). The sample preparation comprised a concentration step and the absolute limits of quantitation were, therefore, 4.7 nmol/l, 18.8 nmol/l, and 0.94 nmol/l for daidzein and genistein, and 9.4 nmol/l, 37.5 nmol/l, and 1.9 nmol/l for equol in urine, serum, and breast tissue homogenate, respectively. Recoveries were between 70% (+/-5.6%) in breast tissue homogenate and 100% (+/-14.1%) in urine and serum for all three compounds. Equol (less than 1 micromol/l homogenate) was found to be the predominant phytoestrogen in breast tissue and its concentrations exceeded those in serum. The concentrations of phytoestrogens were at least 100-fold higher in urine than in serum and breast tissue.     

Chiral separation and determination of R-(-)- and S-(+)-baclofen in human plasma by high-performance liquid chromatography

The method presented here is a high-performance liquid chromatography (HPLC)-UV detection method for the determination of baclofen R-(-)- and S-(+)-enantiomers in human plasma using a chiral separation technique. Baclofen enantiomers were extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge. The extract was then injected onto a HPLC system with a UV detection system set at 220 nm. The separation was achieved by using a 150x4.6 mm, 5 microm Phenomenex chirex 3216 chiral column with a mobile phase consisting of 0.4 mM CuSO(4) in acetonitrile-20 mM sodium acetate (17:83). The calibration curves were linear for both R-(-)- and S-(+)-enantiomers of baclofen in the concentration range of 20-5000 ng/ml. The average regressions were 0.9980 and 0.9991 for R-(-)- and S-(+)-baclofen, respectively. Inter-day precision was 3.3-5.2% for R-(-)-baclofen and 3.5-3.9% for S-(+)-baclofen at a concentration range of 60-4000 ng/ml. Intra-day precisions were 0.6-4.4 and 0.5-3.5% for R-(-)-baclofen and S-(+)-baclofen, respectively. The average extraction recovery was 81.6% for R-(-)-baclofen, 83.0% for S-(+)-baclofen and 94.0% for the internal standard (p-aminobenzoic acid). The limit of quantitation for both R-(-)- and S-(+)-baclofen in human plasma was 20 ng/ml. The method is simple and easy to operate with accuracy and reproducibility and it is suitable for pharmacokinetic studies.     

Receptor binding and biological potency of despentapeptide insulin

The receptor binding and biological potency of despentapeptide insulin (DPI) was assessed in human adipocytes, rat adipocytes and rat hepatocytes. DPI displayed a lower affinity for binding to both human adipocytes (half-maximum displacement at 0.89 +/- 0.04 and 0.20 +/- 0.02 nmol/l for DPI and insulin respectively; P less than 0.001) and rat adipocytes (half-maximum displacement at 7.12 +/- 1.06 and 1.14 +/- 0.18 nmol/l respectively, P less than 0.05). However, although DPI was less potent than unmodified insulin in stimulating glucose uptake in rat adipocytes (half-maximal stimulation at 2.0 +/- 0.67 and 0.47 +/- 0.18 nmol/l respectively; P less than 0.05), DPI was equipotent with insulin in human adipocytes (half-maximal stimulation at 0.034 +/- 0.001 and 0.027 +/- 0.001 nmol/l respectively; P greater than 0.2). In rat hepatocytes, DPI was twofold less potent in binding displacement activity (half-maximum displacement at 3.8 +/- 0.9 and 1.7 +/- 0.3 nmol/l respectively; P less than 0.01) but appeared to be equivalent in stimulating amino butyric acid uptake (half-maximum stimulation at 0.98 +/- 0.12 and 0.95 +/- 0.26 nmol/l respectively). The difference in affinity of DPI binding to rat liver membranes was less marked (1.3 fold decreased compared with insulin: 5.3 +/- 0.7 and 4.2 +/- 0.6 nmol/l respectively; P less than 0.001). Thus, the decreased receptor affinity of DPI was reflected in decreased biological potency in rat adipocytes, but not in human adipocytes nor rat hepatocytes. These data suggest differences in the binding-action linking in the cells of different tissues and different species.     

Markers of acute stress in pigs

This study explores the biological validation of markers of acute stress in pigs subjected to transportation for slaughter. The stress markers selected for monitoring were neopterin and cortisol. Their levels in pig serum were measured for two porcine stress syndrome genotypes, NN and Nn, after a 30-min transport to a slaughterhouse. Blood samples were withdrawn before transport (control group) and immediately after the animals' arrival (experimental group). The values of neopterin and cortisol measured before the transport were 5.60+/-1.65 nmol/l and 273.54+/-66.17 nmol/l respectively. After the transfer, the concentration of cortisol rose significantly compared to the control (355.69+/-85.13 nmol/l, p<0.01). Neopterin concentrations in the serum (8.25+/-1.60 nmol/l) were also significantly higher (p<0.01) after transportation. The elevated concentrations of both analytes were found to be independent of the genotype. These results document the stimulation of the endocrine system and the immune system that develops in animals undergoing transportation for slaughter.     

3种菊花病毒/类病毒实时荧光定量RT-PCR检测体系的构建与准确性评价

菊花容易受到病毒感染而造成品质下降,目前国内对菊花病毒的检测主要根据外观表现或者定性PCR检测,无法准确判定病毒载量。为构建一种可同时用于检测菊花B病毒(Chrysanthemum virus B,CVB)、番茄不孕病毒(Tomato aspermy virus,TAV)和菊花褪绿斑驳类病毒(Chrysanthemum chloritic mottle viroid,CChMVd)的实时荧光定量RT-PCR检测方法,本研究分别以保守区域作为靶标设计相应的引物探针,通过优化扩增体系中CVB、TAV、CChMVd 3种病毒/类病毒探针浓度、引物浓度、Mg²⁺浓度、dNTPs浓度,摸索扩增程序中反转录时间、退火温度和扩增循环数,构建了一种可同时用于CVB、TAV、CChMVd的3重实时荧光定量RT-PCR检测体系,优化后的扩扩增体系中CVB、TAV和CChMVd的探针浓度分别为100 nmol/L、120 nmol/L和80 nmol/L,引物浓度分别为200 nmol/L、240 nmol/L和160 nmol/L,Mg²⁺浓度为3.0 mmol/L;dNTPs浓度200μmol/L;最适反转录时间为25 min,退火温度为60℃,循环数为40。敏感性实验结果表明,该反应体系对3种病毒/类病毒的敏感性为1.0×¹⁰3拷贝/mL,敏感性好;定量线性范围为1.0×¹⁰3拷贝/mL~1.0×¹⁰10拷贝/mL,线性范围宽;特异性好,对菊花矮化类病毒、烟草花叶病毒和黄瓜花叶病毒核酸检测结果为阴性;对1.0×¹⁰4拷贝/mL的低浓度参考品平行检测10次,定量结果lg值偏差(CV%)为4.81%,重复性好。在南京农业大学"中国菊花种质资源保存中心"基地随机选择菊花20株进行本研究试剂检测,检出6例CVB病毒株和4例TAV病毒株,其病毒载量为2.5×¹⁰4拷贝/mL~5.5×¹⁰7拷贝/mL,随机选择1株CVB病毒株定量PCR,产物进行TA克隆后经测序与NCBI Blast比对,其与MH678704.1的同源性为100%。因此,本研究建立了一种能同时检测CVB、TAV、CChMVd 3种菊花常见病毒/类病毒的灵敏、快速、可定量的检测方法。     

Direct chemiluminescence immunoassay (CLIA) for muramyl tripeptide phosphatidyl-ethanolamine in plasma

A competitive chemiluminescent immunoassay for quantitation of muramyl tripeptide phosphatidyl-ethanolamine (MTP-PE) in plasma has been developed. The assay is based on the use of an acridinium ester-labelled analogue of muramyl tripeptide and a rabbit antiserum. It includes an overnight incubation and a separation with a second antibody covalently coupled to paramagnetic particles. The sensitivity of detection is 0.012 nmol/l, the assay working range is 0.1-5 nmol/l, and the inter-assay CVs are ? 10%. Using up to 6000-fold sample dilutions, a wide working range (0.1-30 000 nmol/l) is obtained. Rat plasma samples were collected during and one day after intravenous infusion of MTP-PE. Following infusion, the concentrations in plasma declined multiphasically. Half-life time was 0.37 h ± 0.03 (mean ± SD, alpha phase) and 1.76 h ± 0.08 (mean ± SD, beta phase), clearance and volume of distribution were 0.09 ± 0.02 l/h × kg (mean ± SD) and 0.06 ± 0.01 l/kg (mean ± SD) respectively. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the problems associated with reagents of limited shelf-life.     

Comparative study of semi-specific Aeromonas hydrophila and universal Pseudomonas fluorescens biosensors for BOD measurements in meat industry wastewaters

Aeromonas hydrophila P69.1 (A. hydrophila) was used to construct a semi-specific biosensor to estimate biochemical oxygen demand (BOD) in high fat and grease content wastewaters. A. hydrophila cells were grown in fat containing medium to induce necessary enzymes for transport and degradation of fatty substances. Universal biosensor based on non-specific Pseudomonas fluorescens P75 (P. fluorescens) was used to conduct comparison experiments. Biosensors were calibrated using OECD synthetic wastewater and steady-state method, subsequently several experiments with synthetic and industrial wastewaters were conducted. A linear range up to 45 mg l(-1) BOD(7) was gained using A. hydrophila biosensor, in comparison to 40 mg l(-1) BOD(7) obtained using P. fluorescens biosensors. The lower limit of detection was 5 mg l(-1) BOD(7). Service life of A. hydrophila and P. fluorescens biosensors were 110 and 115 days, respectively. The response time of the biosensors depended on the BOD(7) of measuring solution and was up to 20 min when analyzing different wastewaters. Both biosensors underestimated BOD in meat industry wastewater from 43% up to 71%, but more accurate results could be obtained with A. hydrophila biosensor. Semi-specific A. hydrophila biosensor was able to measure proportion of fat found in wastewater sample, while other refractory compounds remained undetectable to both biosensors.