Facile preparation of a cost-effective platform based on ZnFe2O4 nanomaterials for electrochemical cell detection.

Circulating tumor cells (CTCs) are important tumor markers that indicate early metastasis, tumor recurrence, and treatment efficacy. To identify and separate these cells from the blood, new nanomaterials need to be developed. The present study explored the potential application of ZnFe2O4 magnetic nanoparticles in capturing CTCs with cell surface markers. Folic acid was coupled to L-cysteine-capped ZnFe2O4 nanoparticles (ZC) to provide binding sites on ZnFe2O4 nanoparticles for the recognition of folate bioreceptors, which are highly expressed in MCF-7 breast cancer cells. The cytotoxicity of ZnFe2O4 nanoparticles and ZC against MCF-7 was analyzed with the MTT assay. After 24 h of incubation, there were IC50 values of 702.6 and 805.5 µg/mL for ZnFe2O4 and ZC, respectively. However, after 48 h of incubation, IC50 values of ZnFe2O4 and ZC were reduced to 267.3 and 389.7 µg/mL, respectively. The cell quantification was conducted with magnetically collected cells placed on a glassy carbon electrode, and the differential pulse voltammetry (DPV) responses were analyzed. This cost-effective ZnFe2O4-based biosensing platform allowed cancer cell detection with a limit of detection of 3 cells/mL, ranging from 25 to 104 cells/mL. In future, these functionalized zinc ferrites may be used in electrochemical cell detection and targeted cancer therapy.

Hierarchal polyaniline-folic acid nanostructures act as a platform for electrochemical detection of tumor cells.

The fabrication of electrochemical sensing platforms for cancer monitoring by quantifying circulating tumor cells (CTCs) in blood holds promise for providing a low-cost, rapid, feasible, and safe approach for cancer diagnosis. Here, we isolate cancer cells using CoFe2O4 nanoparticles functionalized with folic acid and chitosan as an inexpensive magnetic nanoprobe. This electrochemical cytosensing platform was realized using polyaniline-folic acid nanohybrids with a three-dimensional hierarchical structure that presents abundant affinity sites toward overexpressed folate bioreceptors on cancer cells, in addition to retaining satisfied conductivity. Furthermore, 3D modeling and simulation of the polyaniline-folic acid structures were conducted to investigate the stable complex between aniline and folate, and the interaction between the polyaniline-folate complex and folate receptor alpha1, a bioreceptor on MCF-7 was revealed for the first time. The limit of detection was calculated to be 4 cells mL-1 with a linear range from 50 to 106 cells mL-1.

Epigenetic Dysregulation of BRDT Gene in Testis Tissues of Infertile Men: Case-Control Study.

Objective: Bromodomain testis associated (BRDT), a testis-specific member of the Bromo- and Extra-Terrminal domain (BET) protein family, is involved in spermatogenesis and, more specifically, chromatin remodeling. In the post-meiotic spermatogenic cells, BRDT protein binds to the hyperacetylated histones and facilitates their replacement with transition proteins (TPs), particularly protamines, which are essential for chromatin condensation. The current research was conducted to assess the expression and epigenetic profile of BRDT in the testis tissues of infertile men. Materials and Methods: In this case-control study, three groups were included: positive control group: obstructive azoospermia (OA, n=10), round spermatid maturation arrest group (SMA, n=10) and negative control group: sertoli cellonly syndrome (SCOS, n=10). Using quantitative real-time polymerase chain reaction (PCR), the expression profile of BRDT was generated. Also, ChIP-real time PCR was used to measure the following histone marks: H3K9ac, H3K9me3, H3K4me3, H3K27me3 on the promoter region of BRDT. Results: Our data indicated that BRDT expression decreased in the SMA group in comparison with the positive control group and this finding is in line with the ChIP results obtained in this group. Conclusion: Based on these data, we postulate that BRDT gene has a vital role in the spermatogenesis and its decreased expression due to an aberrant epigenetic signaling might be associated with male infertility.

MXene-based cytosensor for the detection of HER2-positive cancer cells using CoFe2O4@Ag magnetic nanohybrids conjugated to the HB5 aptamer.

MXenes are a new class of conductive two-dimensional material which have received growing attention in biosensing for their significant surface area and unique surface chemistry. Here, gold electrodes were modified with MXene nanosheets of about 2 nm thickness and 1.5 µm lateral size for the electrochemical detection of tumor cells. An HB5 aptamer with high selectivity for HER-2 positive cells was immobilized on the MXene layers via electrostatic interactions. To minimize electrode biofouling with blood matrix, magnetic separation of HER-2 positive circulating tumor cells was carried out using CoFe2O4@Ag magnetic nanohybrids bonded to the HB5. The formation of sandwich-like structures between the magnetically captured cells and the functionalized MXene electrodes effectively shields the electron transfer of a redox probe, enabling quantitative cell detection using the change in current. This label-free MXene-based cytosensor platform yielded a wide linear range of 102-106 cells/mL, low detection limit of 47 cells/mL, and good sensitivity and selectivity in the detection of HER2-posetive cells in blood samples. The presented aptacytosensor demonstrates the great potential of using CoFe2O4@Ag magnetic nanohybrids and MXenes to monitor cancer progression via circulating tumor cells in blood at low cost.

Relationship between Serum Tumor-Related Markers and Genetically Modified Rice Expressing Cry1Ab Protein in Sprague-Dawley Rats.

Genetically Modified (GM) foods are among the most important achievements of biotechnology. Given the safety importance of transgenic rice, this study was conducted to investigate the effect of GM rice consumption on serum concentrations of tumor markers in rats. In this experimental intervention, we used the blood serum samples from the Biobank taken from 60 males and 60 female Sprague-Dawley (SD) rats fed on three different diets, including rat's standard food, non-GM rice, and GM rice after 90 day. Tumor markers including Carcinogenic embryonic antigen (CEA), Alpha-Fito protein (AFP), Cancer Antigen 19-9 (CA19-9), Cancer Antigen 125 (CA125), and Cancer Antigen15-3 (CA15-3) were assessed by enzyme-linked immune sorbent assay (ELISA) method. Statistical analysis was conducted via SPSS software. The results show that the concentrations of tumor markers were within the normal range in the SD rats treated with diet, and since the concentration of tumor markers was lower than the acceptable index determined, according to the kit standard in both groups, no obvious carcinogenic effect was found. However, these findings are not enough to make a final decision regarding the safety assessment of GM rice consumption.

Evaluation the Presence of SERPINA5 (Exon 3) and FTO rs9939609 Polymorphisms in Papillary Thyroid Cancer Patients.

BACKGROUND: A few researches evaluated the association of polymorphisms at SERPINA5 and fat mass and obesity-associated protein (FTO) genes with papillary thyroid cancer (PTC) globally. Here, we examined the presence of genetic variations within coding exon 3 of SERPINA5 gene and FTO rs9939609 polymorphism in Iranian PTC patients. METHODS: A total of 122 patients (42 cases for SERPINA5 and 80 cases for FTO gene) and 120 healthy subjects (40 subjects or SERPINA5 and 80 subjects for FTO gene) were recruited. The genetic variation within coding exon 3 of SERPINA5 gene was evaluated by reaction-single-strand conformation polymorphism (PCR-SSCP) and FTO rs9939609 polymorphism was evaluated by RFLP-PCR assay. RESULTS: The PCR-SSCP technique detected two rs6115G>A and rs6112T>C genetic variations within coding exon 3 of SERPINA5 gene and approved also by direct sequencing. For rs6112T>C polymorphism seven patients was heterozygous and for rs6115G>A seven PTC patients were heterozygous and two patients were homozygous. CONCLUSION: This study indicated that SERPINA5 rs6115G>A and rs6112T>C polymorphisms might be a novel susceptibility locus for PTC in Iranian patients. However, our findings do not support an association between FTO rs9939609 polymorphism and PTC risk.

In situ monitoring of gating approach on mesoporous silica nanoparticles thin-film generated by the EASA method for electrochemical detection of insulin.

An innovative label-free electrochemical aptasensing platform has been designed for detection of insulin using functionalized mesoporous silica thin-film (MSTF) coated on a glassy carbon electrode through the one-step electrochemically assisted self-assembly (EASA) method. This strategy is contingent upon the covalent attachment of a complementary DNA (cDNA) oligonucleotide sequence on the mesoporous silica surface, for which further hybridization with its labeled aptamer as a gating molecule restricts the diffusion of the electroactive probe (Fe(CN)63-/4-) toward the electrode surface by the closing of mesochannels. Upon insulin introduction as the stimulus target molecule, hybridization between aptamer and cDNA is efficiently destroyed, which triggers the opening of nanochannels to facilitate redox probe diffusion toward the electrode with a noticeable increase in differential pulse voltammetry signal. The proposed aptasensor showed a wide detection ranging from 10.0 to 350.0 nM and a suitable detection limit of 3.0 nM. This method offers the sensitive and rapid detection of insulin without the need for cargo (dye/fluorophore) as an electrochemical marker inside the pore, at low cost and with a fast modification time.

Safety assessment of genetically modified rice expressing Cry1Ab protein in Sprague-Dawley rats.

Rice is considered one of the most important staple food crops. Genetically modified (GM) Bt rice, harbored cry1Ab gene expressing the insect-resistance protein has been developed to resistance to the insects. In this study, we assessed the safety of the GM Bt rice on Sprague-Dawley rats for 90 days. Totally, 120 rats in both sexes were used for three different diets, including 50% GM Bt rice, feeding with 50% rice, and standard feeding. Each 40 SD rats including 20 males and 20 females were considered as each diet. The clinical variables such as body weight and food consumption were measured and a range of clinical tests was examined, including hematology, serum chemistry parameters, urinalysis profile, thyroid, and sex hormone levels. Pathological assessments were also done. The results showed that the mean weekly feed utilization (%) had no significant difference among the studied groups. Also, blood biochemistry, hematological parameters, urine analysis, and hormonal levels had no significant differences among the groups. However, alanine aminotransferase was less in males versus female feeding with GM Bt rice. No histopathological changes were observed among the groups. In conclusion, this study demonstrated that GM Bt rice had no obvious adverse effects on rats' health.

Designing and optimization of an electrochemical substitute for the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay.

For the first time ever, this paper reports the development of an easily operated and cost-effective electrochemical assay to be used as an appropriate substitute for the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay. The proposed assay is based on the electrochemical reaction of Saccharomyces cerevisiae (S. cerevisiae) with toxic materials, and it overcomes most of the limitations of MTT such as evaporation of volatile solvents, cytotoxic effects of MTT reagents, high cost, and sensitivity to light. The novel electrochemical assay can be used to detect diazinon in the range of 10-6 g mL-1 to 10-2 g mL-1 with the detection limit of 1.5 × 10-7 g mL-1.

Protein tertiary structure prediction using hidden Markov model based on lattice.

The prediction of protein structure from its amino acid sequence is one of the most prominent problems in computational biology. The biological function of a protein depends on its tertiary structure which is determined by its amino acid sequence via the process of protein folding. We propose a novel fold recognition method for protein tertiary structure prediction based on a hidden Markov model and 3D coordinates of amino acid residues. The method introduces states based on the basis vectors in Bravais cubic lattices to learn the path of amino acids of the proteins of each fold. Three hidden Markov models are considered based on simple cubic, body-centered cubic (BCC) and face-centered cubic (FCC) lattices. A 10-fold cross validation was performed on a set of 42 fold SCOP dataset. The proposed composite methodology is compared to fold recognition methods which have HMM as base of their algorithms having approaches on only amino acid sequence or secondary structure. The accuracy of proposed model based on face-centered cubic lattices is quite better in comparison with SAM, 3-HMM optimized and Markov chain optimized in overall experiment. The huge data of 3D space help the model to have greater performance in comparison to methods which use only primary structures or only secondary structures.

Fabrication of an ultrasensitive and selective electrochemical aptasensor to detect carcinoembryonic antigen by using a new nanocomposite.

A lable-free electrochemical aptasensor was successfully developed for the sensitive detection of carcinoembryonic antigen as a tumor biomarker. To do this, a ternary nanocomposite of hemin, graphene oxide and multi-walled carbon nanotubes was used. The aptamer can be attached to the surface of a hemin, graphene oxide and multi-walled carbon nanotubes glassy carbon electrode through -NHCO- covalent bonds to form a sensing surface. Through fourier transform infrared spectroscopy and scanning electron microscopy, it was indicated that hemin can be successfully incorporated into hemin, graphene oxide and multi-walled carbon nanotubes. Hemin, which protects graphene nanosheets, also serves as an in-situ probe owing to its well-defined redox properties. Multi-walled carbon nanotubes in the modifier enhance conductivity and facilitate the electron transfer between hemin and the glassy carbon electrode. In this study, carcinoembryonic antigen got specifically bound to the aptamer, and the current changes were used for selective and specific detection of that antigen. The devised aptasensor proved to have excellent performance with a wide linear range of 1.0 × 10-15 - 1.0 × 10-8 gmL-1 and a detection limit of 0.82 fg mL-1. The inter-day and intra-day values of RSD% were obtained in the range of 0.10-2.91 and 2.21-4.56 respectively. According to the experiments conducted on real samples, it may be claimed that the proposed label-free electrochemical aptasensor is capable enough of determining carcinoembryonic antigen in clinical diagnostics.

A Composite Approach to Protein Tertiary Structure Prediction: Hidden Markov Model Based on Lattice.

The biological function of protein depends mainly on its tertiary structure which is determined by its amino acid sequence via the process of protein folding. Prediction of protein structure from its amino acid sequence is one of the most prominent problems in computational biology. Two basic methodologies on protein structure prediction are combined: ab initio method (3-D space lattice) and fold recognition method (hidden Markov model). The primary structure of proteins and 3-D coordinates of amino acid residues are put together in one hidden Markov model to learn the path of amino acid residues in 3-D space from the first atom to the last atom of each protein of each fold. Therefore, each model has the information of 3-D path of amino acids of each fold. The proposed method is compared to fold recognition methods which have hidden Markov model as a base of their algorithms having approaches on only amino acid sequence or secondary structure. To validate the proposed method, the models are assessed with three datasets. Results show that the proposed models outperform 7-HMM and 3-HMM in the same dataset. The face-centered cubic lattice which is the most compacted 3-D lattice reached the maximum classification accuracy in all experiments in comparison with the performance of the most effective version of optimized 3-HMM as well as the performance of the latest version of SAM 3.5. Results show that 3-D coordinates of atoms of amino acids in proteins have an important role in prediction. It also has great hidden information as compared to secondary structure of proteins in fold classification.

Molecular Diagnosis of Bacterial Definite Infective Endocarditis by Real-Time Polymerase Chain Reaction.

Infective endocarditis (IE) can be diagnosed using the Duke criteria, which cannot be conclusive especially when the results of blood cultures are negative. This study aimed at using real-time polymerase chain reaction (PCR) technique to isolate bacteria present in whole blood samples of patients with definitive IE on the basis of the method designed in this study. This laboratory and test study was conducted on 20 whole blood samples taken from patients with definitive IE. Real-time PCR of the 16s rRNA was utilized to directly analyze whole blood samples to diagnose bacterial IE. Of 20 whole blood samples with definitive IE, only one blood culture (5%) was positive and the isolated bacterium belonged to Streptococci viridans group. Also, 13 whole blood samples were positive using real-time PCR technique. The isolated bacteria were Enterococcus faecalis with seven (35%) cases, Streptococcus gallolyticus with two (10%) cases, Streptococcus mutans with one (5%) case, Streptococcus sanguinis with one (5%) case, Streptococcus salivarius with one (5%) case, and Staphylococcus aureus with one (5%) case. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) using real-time PCR technique were 65%, 100%, 100%, and 74%, respectively. The developed real-time PCR method allows us to detect bacteria in whole blood samples and is much more sensitive than culturing method. It also permits the differentiation of the main group of bacteria within a few hours for IE.

The diagnosis of microorganism involved in infective endocarditis (IE) by polymerase chain reaction (PCR) and real-time PCR: A systematic review.

Broad-range bacterial rDNA polymerase chain reaction (PCR) followed by sequencing may be identified as the etiology of infective endocarditis (IE) from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery.

Evaluation of epidemiological, clinical, and microbiological features of definite infective endocarditis.

Background: Infective endocarditis (IE) is a microbial infection of heart valves and its endothelial lining which is considered as a life-threatening disorder. This study evaluated the epidemiological, clinical, and microbiological features of IE at the Cardiovascular Research Center in Yazd, Iran. Methods: The cross-sectional study was conducted on 20 patients diagnosed with definite IE on the basis of Duke's criteria hospitalized for one year in the Cardiovascular Research Center in Yazd, Iran, from January 2015 to December 2015. Demographic information, clinical, laboratory, and microbiological findings, and also trans-esophageal echocardiography (TEE) of each patient were recorded and assessed. The collected data were analyzed using SPSS 16. Results: The mean age of the patients under study was 45±16 years with most of the afflicted patients (60%) being male. Most cases (70%) of IE were observed in the warm seasons (spring and summer). The most common clinical sign (80%) was fever. TEE was positive for all (100%) patients, and vegetation was seen in all patients. The nosocomial mortality rate was zero. However, 14 (70%) patients underwent surgical treatment. The valves afflicted with IE were: the mitral valve (40%), the aortic valve (35%), and the tricuspid valve (25%), respectively. 4 patients (20%) had a positive history of IE. Blood culture test was positive only in 1 case and the isolated microorganism belonged to the viridans group streptococci. Conclusion: Despite the one-year high prevalence of IE in this study, the nosocomial mortality rate was not high and was reported to be nil under surgical and antimicrobial therapy.

Novel Mutations of the CHRNA3 Gene in Non-Small Cell Lung Cancer in an Iranian Population

Background: Lung cancer, the leading cause of cancer-related worldwide deaths, largely results from the combined effects of smoking exposure and genetic susceptibility. CHRNA3, a nicotinic acetylcholine receptor gene, is associated with lung cancer risk. This study sought to identify variations in exon 3 of CHRNA3 in an Iranian population with non-small cell lung cancer (NSCLC). Materials and methods: A case-control study including 147 individuals with lung cancer and 145 healthy individuals was conducted. As mobility shift caused by nucleotide substitutions might be due to a conformational change of single-stranded DNAs, we designated these as single-strand conformation polymorphisms (SSCPs). PCR amplified products with SSCP were subjected to DNA sequencing. Results: The sequencing results showed 3 polymorphisms in exon 3 of CHRNA3, rs8040868, rs763384023 and rs2869547 , the latter two of which have not been reported in NSCLC, previously. Conclusion: It appears that the rs8040868 may be considered as a pathogenic mutation associated with the clinical phenotype. Polymorphisms are important factors for development of cancers and may provide additional insights into mechanisms underlying NSCLC.

Haplotype analysis of BRCA1 intragenic markers in Iranian patients with familial breast and ovarian cancer.

BACKGROUND: Breast cancer is the most common malignancy in women. Breast Cancer Type 1 Susceptibility gene (BRCA1) is a tumor suppressor gene, involved in DNA damage repair and in 81% of the breast-ovarian cancer families were due to BRCA1. In some clinically investigated genes, the intragenic marker polymorphism is important and the screening of such mutations is faster by using short tandem repeat (STR) polymorphism. Individual polymorphism of STR is a good evidence for following inheritance of repeat polymorphism. OBJECTIVE: The aim of this study was to evaluate three intragenic BRCA1 marker polymorphisms in families, which have two or more patients with breast/ovarian cancer in comparison to healthy women. MATERIALS AND METHODS: A total of 107 breast and/or ovarian cancer patients and 93 unrelated healthy women with no clinical phenotype of any malignancy or familial cancer history constitute the study groups. Haplotyping analysis, at 3 intragenic BRCA1 microsatellite markers (D17S855, D17S1322 and D17S1323), were performed for all subject and control groups using labeled primers. RESULTS: After fragment analysis, significance differences were observed as follows: two alleles of D17S855; allele 146 (p=0.02) and 150 (p=0.006), and two alleles of D17S1322, allele 121 (p=0.015) and 142 (p=0.043). These differences were compared with control group. There was significance difference in 8 di/tri allelic haplotypes in present experimental subjects. Some haplotypes were observed to have approximately twice the relation risk for breast cancer. CONCLUSION: According to recent results, assessment of presence or absence of mentioned alleles in BRCA1 microsatellite can be used for prognosis in individuals, suspected of having or not having the breast cancer.

Comparison of impedimetric detection of DNA hybridization on the various biosensors based on modified glassy carbon electrodes with PANHS and nanomaterials of RGO and MWCNTs.

In this research, we have developed lable free DNA biosensors based on modified glassy carbon electrodes (GCE) with reduced graphene oxide (RGO) and carbon nanotubes (MWCNTs) for detection of DNA sequences. This paper compares the detection of BRCA1 5382insC mutation using independent glassy carbon electrodes (GCE) modified with RGO and MWCNTs. A probe (BRCA1 5382insC mutation detection (ssDNA)) was then immobilized on the modified electrodes for a specific time. The immobilization of the probe and its hybridization with the target DNA (Complementary DNA) were performed under optimum conditions using different electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The proposed biosensors were used for determination of complementary DNA sequences. The non-modified DNA biosensor (1-pyrenebutyric acid-N- hydroxysuccinimide ester (PANHS)/GCE), revealed a linear relationship between ∆Rct and logarithm of the complementary target DNA concentration ranging from 1.0×10(-16)molL(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.992, for DNA biosensors modified with multi-wall carbon nanotubes (MWCNTs) and reduced graphene oxide (RGO) wider linear range and lower detection limit were obtained. For ssDNA/PANHS/MWCNTs/GCE a linear range 1.0×10(-17)mol L(-1)-1.0×10(-10)mol L(-1) with a correlation coefficient of 0.993 and for ssDNA/PANHS/RGO/GCE a linear range from 1.0×10(-18)mol L(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.985 were obtained. In addition, the mentioned biosensors were satisfactorily applied for discriminating of complementary sequences from noncomplementary sequences, so the mentioned biosensors can be used for the detection of BRCA1-associated breast cancer.

Direct spectrophotometric assay of laccase using diazo derivatives of guaiacol.

Laccase (EC 1.10.3.2) is a widespread cuproenzyme able to oxidize various types of phenols and similar aromatic compounds through a one-electron transfer mechanism. The enzyme has already found its way into the market as a biocatalyst. Because of its ability to be paired by electron mediators, the expectation for employing laccases in versatile processes is very high. There are a few spectrophotometric methods for assaying the laccase activity; however, all of them are based on the formation of product(s) resulting from the enzymatic and inevitable succeeding chemical reactions. Use of diazo derivatives of guaiacol (DdG) was developed as a new spectrophotometric method based on substrate depletion allowing direct assessment of enzyme activity has been introduced. This method allows accurate comprehensive kinetic studies of laccases and provides reliable information about the quality of docking of different substrates or one substrate to the active sites of different laccases. Using this method, the kinetic parameters of various DdG carrying different electron donating and withdrawing substituents were used to assay laccase from Neurospora crassa. 2-Methoxy-4-[(4-phenyl)azo]-phenol (K(m) = 93.5 µM and V = 1.98 µM/min) was identified as an appropriate substrate for the accurate and routine spectrophotometric based assay of laccases.