Application of zeolites and zeolitic imidazolate frameworks in the biosensor development.

Biosensors are advanced devices for analysis of composition of blood, urine, environmental samples, and many other media. Their current development is tightly linked with nanomaterials, such as zeolites and zeolitic imidazolate framework (ZIFs). The present review describes electrochemical (amperometric, conductometric, ISFET) and optical (fluorescent and colorimetric) biosensors that incorporate zeolites and ZIFs in their biorecognition elements. The biosensors are based on immobilized enzymes (such as glucose oxidase, urease, and acetylcholinesterase), antibodies, DNA, and aptamers. The review present reasons for application of these nanomaterials, and discusses advantages of zeolite- and ZIF-containing biosensors over other biosensors. In most cases, the biosensors have improved sensitivity, better limit of detection, wider linear range, and other improved characteristics. It is demonstrated that immobilization of biomolecules such as enzymes or antibodies on the surface of zeolites and ZIFs enables creation of unique advanced biosensors that have a potential for further development and practical applications.

Electrochemical biosensors based on multienzyme systems: Main groups, advantages and limitations - A review.

In the review, the principles and main purposes of using multienzyme systems in electrochemical biosensors are analyzed. Coupling several enzymes allows an extension of the spectrum of detectable substances, an increase in the biosensor sensitivity (in some cases, by several orders of magnitude), and an improvement of the biosensor selectivity, as showed on the examples of amperometric, potentiometric, and conductometric biosensors. The biosensors based on cascade, cyclic and competitive enzyme systems are described alongside principles of function, advantages, disadvantages and practical use for real sample analyses in various application areas (food production and quality control, clinical diagnostics, environmental monitoring). The complications and restrictions regarding the development of multienzyme biosensors are evaluated. The recommendations on the reasonability of elaboration of novel multienzyme biosensors are given.

A highly selective amperometric biosensor array for the simultaneous determination of glutamate, glucose, choline, acetylcholine, lactate and pyruvate.

The work was aimed at the development of a biosensor array for the simultaneous determination of six solutes (glutamate, glucose, choline, acetylcholine, lactate, and pyruvate) in aqueous solutions. Enzymes selective for these substrates were immobilized on the surface of amperometric platinum disc electrodes and served as bioselective elements of a biosensor array. Direct enzymatic analysis by the developed biosensors provided high sensitivity to the tested substrates (limits of detection were 1-5 µM). The linear ranges of the biosensors were from 0.001-0.01 mM to 0.2-2.5 mM. The influence of solution pH, ionic strength and buffer capacity on the biosensor responses was investigated; the conditions for simultaneous operation of all the bioselective elements were optimized. The absence of any cross-influence of the substrates of enzymatic systems used was shown as well as a high selectivity of the biosensors and the absence of any impact of interfering substances (ascorbic acid, dopamine, cysteine, paracetamol). The developed biosensor array had good response reproducibility and storage stability. The array is suitable for rapid (0.5-1 min) and simple simultaneous determination of glutamate, glucose, choline, acetylcholine, lactate, and pyruvate in aqueous (biological) samples; furthermore, the creation of a single chip with six sensitive elements is possible as well as the addition of other biosensors.

An amperometric glutamate biosensor for monitoring glutamate release from brain nerve terminals and in blood plasma.

An excess of the excitatory neurotransmitter, glutamate, in the synaptic cleft during hypoxia/ischemia provokes development of neurotoxicity and originates from the reversal of Na+-dependent glutamate transporters located in the plasma membrane of presynaptic brain nerve terminals. Here, we have optimized an electrochemical glutamate biosensor using glutamate oxidase and developed a biosensor-based methodological approach for analysis of rates of tonic, exocytotic and transporter-mediated glutamate release from isolated rat brain nerve terminals (synaptosomes). Changes in the extracellular glutamate concentrations from 11.5 ±â€¯0.9 to 11.7 ±â€¯0.9 µΜ for 6 min reflected a low tonic release of endogenous glutamate from nerve terminals. Depolarization-induced exocytotic release of endogenous glutamate was equal to 7.5 ±â€¯1.0 µΜ and transporter reversal was 8.0 ±â€¯1.0 µΜ for 6 min. The biosensor data correlated well with the results obtained using radiolabelled L-[14C]glutamate, spectrofluorimetric glutamate dehydrogenase and amino acid analyzer assays. The blood plasma glutamate concentration was also tested, and reliability of the biosensor measurements was confirmed by glutamate dehydrogenase assay. Therefore, the biosensor-based approach for accurate monitoring rates of tonic, exocytotic and transporter-mediated release of glutamate in nerve terminals was developed and its adequacy was confirmed by independent analytical methods. The biosensor measurements provided precise data on changes in the concentrations of endogenous glutamate in nerve terminals in response to stimulation. We consider that the glutamate biosensor-based approach can be applied in clinics for neuromonitoring glutamate-related parameters in brain samples, liquids and blood plasma in stroke, brain trauma, therapeutic hypothermia treatment, etc., and also in laboratory work to record glutamate release and uptake kinetics in nerve terminals.

Selective Amplification of SPR Biosensor Signal for Recognition of rpoB Gene Fragments by Use of Gold Nanoparticles Modified by Thiolated DNA.

An experimental approach for improving the sensitivity of the surface plasmon resonance (SPR) DNA hybridization sensor using gold nanoparticles (GNPs), modified by specific oligonucleotides, was elaborated. An influence of the ionic strength on the aggregation stability of unmodified GNPs and GNPs modified by the thiolated oligonucleotides was investigated by monitoring a value of light extinction at 520 nm that can be considered as a measure of a quantity of the non-aggregated GNPs. While the unmodified GNPs started to aggregate in 0.2 × saline-sodium citrate (SSC), GNPs modified by the negatively charged oligonucleotides were more stable at increasing ionic strength up to 0.5 × SSC. A bioselective element of the SPR DNA hybridization sensor was formed by immobilization on the gold sensor surface of the thiolated oligonucleotides P2, the sequence of which is a fragment of the rpoB gene of Mycobacterium tuberculosis. The injections into the measuring flow cell of the SPR spectrometer of various concentrations of GNPs modified by the complementary oligonucleotides T2-18m caused the pronounced concentration-dependent sequence-specific sensor responses. The magnitude of the sensor responses was much higher than in the case of the free standing complementary oligonucleotides. According to the obtained experimental data, the usage of GNPs modified by specific oligonucleotides can amplify the sensor response of the SPR DNA hybridization sensor in ~1200 times.

Surface Plasmon Resonance Investigations of Bioselective Element Based on the Recombinant Protein A for Immunoglobulin Detection.

The developed surface plasmon resonance (SPR) biosensor based on the recombinant Staphylococcal protein A with an additional cysteine residue (SPA-Cys) used as a biorecognition component showed a good selectivity and sensitivity for the immunoglobulin detection. The developed biosensor with SPA-Cys-based bioselective element can also be used as a first step of immunosensor creation. The successful immobilization of SPA-Cys on the nanolayer gold sensor surface of the SPR spectrometer was performed. The efficiency of blocking nonspecific sorption sites on the sensor surface with milk proteins, gelatin, BSA, and HSA was studied, and a rather high efficiency of using gelatin was confirmed. The SPR biosensor selectively interacted with IgG and did not interact with the control proteins. The linear dependence of the sensor response on the IgG concentration in the range from 2 to 10 µg/ml was shown. Using the calibration curve, the IgG concentration was measured in the model samples. The determined concentrations are in good agreement (r 2 = 0.97) with the given concentration of IgG.

A novel conductometric biosensor based on hexokinase for determination of adenosine triphosphate.

The paper presents a simple and inexpensive reusable biosensor for determination of the concentration of adenosine-5'-triphosphate (ATP) in aqueous samples. The biosensor is based on a conductometric transducer which contains two pairs of gold interdigitated electrodes. An enzyme hexokinase was immobilized onto one pair of electrodes, and bovine serum albumin-onto another pair (thus, a differential mode of measurement was used). Conditions of hexokinase immobilization on the transducer by cross-linking via glutaraldehyde were optimized. Influence of experimental conditions (concentration of magnesium ions, ionic strength and concentration of the working buffer) on the biosensor work was studied. The reproducibility of biosensor responses and operational stability of the biosensor were checked during one week. Dry storage at -18 °C was shown to be the best conditions to store the biosensor. The biosensor was successfully applied for measurements of ATP concentration in pharmaceutical samples. The proposed biosensor may be used in future for determination of ATP and/or glucose in water samples.

A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite.

Development of a conductometric biosensor for the urea detection has been reported. It was created using a non-typical method of the recombinant urease immobilization via adsorption on nanoporous particles of silicalite. It should be noted that this biosensor has a number of advantages, such as simple and fast performance, the absence of toxic compounds during biosensor preparation, and high reproducibility (RSD = 5.1 %). The linear range of urea determination by using the biosensor was 0.05-15 mM, and a lower limit of urea detection was 20 µM. The bioselective element was found to be stable for 19 days. The characteristics of recombinant urease-based biomembranes, such as dependence of responses on the protein and ion concentrations, were investigated. It is shown that the developed biosensor can be successfully used for the urea analysis during renal dialysis.

Determination of total creatine kinase activity in blood serum using an amperometric biosensor based on glucose oxidase and hexokinase.

Creatine kinase (CK: adenosine-5-triphosphate-creatine phosphotransferase) is an important enzyme of muscle cells; the presence of a large amount of the enzyme in blood serum is a biomarker of muscular injuries, such as acute myocardial infarction. This work describes a bi-enzyme (glucose oxidase and hexokinase based) biosensor for rapid and convenient determination of CK activity by measuring the rate of ATP production by this enzyme. Simultaneously the biosensor determines glucose concentration in the sample. Platinum disk electrodes were used as amperometric transducers. Glucose oxidase and hexokinase were co-immobilized via cross-linking with BSA by glutaraldehyde and served as a biorecognition element of the biosensor. The biosensor work at different concentrations of CK substrates (ADP and creatine phosphate) was investigated; optimal concentration of ADP was 1mM, and creatine phosphate - 10 mM. The reproducibility of the biosensor responses to glucose, ATP and CK during a day was tested (relative standard deviation of 15 responses to glucose was 2%, to ATP - 6%, to CK - 7-18% depending on concentration of the CK). Total time of CK analysis was 10 min. The measurements of creatine kinase in blood serum samples were carried out (at 20-fold sample dilution). Twentyfold dilution of serum samples was chosen as optimal for CK determination. The biosensor could distinguish healthy and ill people and evaluate the level of CK increase. Thus, the biosensor can be used as a test-system for CK analysis in blood serum or serve as a component of multibiosensors for determination of important blood substances. Determination of activity of other kinases by the developed biosensor is also possible for research purposes.

Development of novel enzyme potentiometric biosensor based on pH-sensitive field-effect transistors for aflatoxin B1 analysis in real samples.

This study aimed at the development and optimization of a potentiometric biosensor based on pH-sensitive field-effect transistors and acetylcholinesterase for aflatoxin B1 determination in real samples. Optimal conditions for bioselective elements operation were defined and analytical characteristics of the proposed biosensor were studied. The proposed biosensor characterized high operational stability and reproducibility of signal. Selectivity of acetylcholinesterase-biosensor to aflatoxins in relation to other groups of toxic substances was analyzed. The developed biosensor was applied to the determination of aflatoxin B1 in real samples (sesame, walnut and pea).

Monitoring of the velocity of high-affinity glutamate uptake by isolated brain nerve terminals using amperometric glutamate biosensor.

Glutamate is the major excitatory neurotransmitter in the central nervous system, which is involved in the main aspects of normal brain functioning. High-affinity Na(+)-dependent glutamate transporters is key proteins, which transport extracellular glutamate to the cytoplasm of nerve cells, thereby preventing continuous activation of glutamate receptors, and thus the development of neurotoxicity. Disturbance in glutamate uptake is involved in the pathogenesis of major neurological disorders. Amperometric biosensors are the most promising and successful among electrochemical biosensors. In this study, we developed (1) amperometric glutamate biosensor, (2) methodological approach for the analysis of glutamate uptake in liquid samples of isolated rat brain nerve terminals (synaptosomes). The basal level of glutamate, the initial velocity of glutamate uptake and time-dependent accumulation of glutamate by synaptosomes were determined using developed glutamate biosensor. Comparative analysis of the data with those obtained by radioactive analysis, spectrofluorimetry and ion exchange chromatography was performed. Therefore, the methodological approach for monitoring of the velocity of glutamate uptake, which takes into consideration the definite level of endogenous glutamate in nerve terminals, was developed using glutamate biosensor.

Feasibility of application of conductometric biosensor based on acetylcholinesterase for the inhibitory analysis of toxic compounds of different nature.

This study was aimed at the development of a conductometric biosensor based on acetylcholinesterase considering the feasibility of its application for the inhibitory analysis of various toxicants. In this paper, the optimum conditions for enzyme immobilization on the transducer surface are selected as well as the optimum concentration of substrate for inhibitory analysis. Sensitivity of the developed biosensor to different classes of toxic compounds (organophosphorus pesticides, heavy metal ions, surfactants, aflatoxin, glycoalkaloids) was tested. It is shown that the developed biosensor can be successfully used for the analysis of pesticides and mycotoxins, as well as for determination of total toxicity of the samples. A new method of biosensor analysis of toxic substances of different classes in complex multicomponent aqueous samples is proposed.

Application of enzyme/zeolite sensor for urea analysis in serum.

Urea biosensor based on zeolite-adsorbed urease was applied for analysis of blood serum samples. It should be noted, that this biosensor has a number of advantages, such as simple and fast performance, the absence of toxic compounds during biosensor preparation, high reproducibility and repeatability (RSD=9% and 4%, respectively). The linear range of urea determination by using the biosensor was 0.003-0.75 mM, and the limit of urea detection was 3 µM. The method of standard addition was used for analysis of serum samples with 500-fold dilution. Total time of analysis was 10 min. Good reproducibility of urea determination in real samples was demonstrated (RSD=10%). Biosensor results were verified by using a common method of urea determination (diacetyl monoxime reaction). It was shown that by using this biosensor distinguishing healthy people from people with renal dysfunction becomes easier.

Development of conductometric biosensor array for simultaneous determination of maltose, lactose, sucrose and glucose.

The aim of this work was to develop an array of biosensors for simultaneous determination of four carbohydrates in solution. Several enzyme systems selective to lactose, maltose, sucrose and glucose were immobilised on the surface of four conductometric transducers and served as bio-recognition elements of the biosensor array. Direct enzyme analysis carried out by the developed biosensors was highly sensitive to the corresponding substrates. The analysis lasted 2 min. The dynamic range of substrate determination extended from 0.001 mM to 1.0-3.0mM, and strongly depended on the enzyme system used. An effect of the solution pH, ionic strength and buffer capacity on the biosensors responses was investigated; the conditions of simultaneous operation of all biosensors were optimised. The data on cross-impact of the substrates of all biosensors were obtained; the biosensor selectivity towards possible interfering carbohydrates was tested. The developed biosensor array showed good signal reproducibility and storage stability. The biosensor array is suited for simultaneous, quick, simple, and selective determination of maltose, lactose, sucrose and glucose.

Discrimination of single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis using a surface plasmon resonance biosensor.

Single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis, the mutations of which cause drug resistance of the infectious agent, were detected and discriminated using a surface plasmon resonance biosensor system. Thiol-modified oligonucleotides of the selected sequence (the probe) and 1-mercapto-6-hexanol were immobilized on a gold sensor surface. Hybridization between immobilized probe P2 and perfectly matched target T2 as well as a single base mismatched target TN was investigated in buffer solutions of various stringencies. Discrimination of perfectly matched and single base mismatched targets is achieved due to a difference in the level of their hybridization with the immobilized probe depending on stringency of the buffer solution. In 0.5×SSC buffer solution (7.5 mM sodium citrate, pH 7, containing 75 mM NaCl), sensor response at T2 injection into the measuring sensor cell was 16 times that at TN injection. The experimental results on surface hybridization between the studied oligonucleotides demonstrated a good correlation with theoretical calculations of thermodynamic parameters of these interactions in the solution. The described approach could be proposed as a basis for creating a biosensor for real-time label-free diagnostics of drug-resistant tuberculosis.

Application of creatinine-sensitive biosensor for hemodialysis control.

The highly sensitive and selective potentiometric biosensor for creatinine determination has been developed by us earlier. In it, pH-sensitive field effect transistors were used as transducer and immobilized creatinine deiminase (EC 3.5.4.21)--as a biosensitive element. In the work presented, we optimized this biosensor for creatinine analysis in real samples of dialysate in patients with renal failure. The optimized version of biosensor was applied for on-line monitoring of the level of creatinine in the patient's dialysate fluid in the course of dialysis session. High correlation between the biosensor analysis and traditional Jaffe method was demonstrated.

Novel conductometric biosensor based on three-enzyme system for selective determination of heavy metal ions.

A differential pair of planar thin-film interdigitated electrodes, deposited on a ceramic pad, was used as a conductometric transducer. The three-enzyme system (invertase, mutarotase, glucose oxidase), immobilized on the transducer surface, was used as a bioselective element. The ratio between enzymes in the membrane was found experimentally considering the highest biosensor sensitivity to substrate (sucrose) and heavy metal ions. Optimal concentration of sucrose for inhibitory analysis was 1.25 mM and incubation time in the investigated solution amounted to 10-20 min. The developed biosensor demonstrated the best sensitivity toward ions Hg(2+) and Ag(+). A principal possibility of the biosensor reactivation either by EDTA solution after inhibition with silver ions or by cysteine solution after inhibition with mercury ions was shown.

Effect of different modifications of BEA-zeolites on operational characteristics of conductometric biosensor.

Effect of different modifications of zeolite Na(+)-BEA on working characteristics of urease-based conductometric biosensor was studied. As the biosensor sensitive elements were used bioselective membranes based on urease and various zeolites immobilised with bovine serum albumin on the surface of conductometric transducers. Influence of zeolites on sensitivity of urea biosensor was investigated as well as reproducibility of biosensor signal and reproducibility of activity of the bioselective element after different variants of urease immobilisation on the surface of conductometric transducer. The biosensors based on zeolites (NH4(+)-BEA 30 and H(+)-BEA 30) were shown to be the most sensitive. Concentration of these zeolites in the bioselective membrane was optimized. Use of zeolites modified with methyl viologen and silver was ascertained to be of no prospect for urea conductometric biosensors. It was demonstrated that characteristics of urea biosensors can be regulated, varying zeolites modifications and their concentrations in bioselective membranes.

Surface plasmon resonance detection of oligonucleotide sequences of the rpoB genes of Mycobacterium tuberculosis.

Oligonucleotide sequences related to the normal and mutated rpoB genes of Mycobacterium tuberculosis are detected using a surface plasmon resonance (SPR) biosensor system. A bioselective element was prepared by immobilizing the thiol-modified oligonucleotides of the selected sequence (the capture probe P2) that contains the mutated TCG→TTG codon 531 (evoking drug resistance) of the rpoB gene of M. tuberculosis on a gold sensor surface. Specific hybridization between immobilized probe P2 and complementary target T2 gave the highest sensor response, single-base mismatched oligonucleotide TN (corresponding to the normal gene sequence) produced somewhat smaller response and no response was observed at injection of noncomplementary oligonucleotide TC. The P2-T2 hybridization efficiency is calculated ca. 30% (5 × 10(12) molecules cm(-2)), and the lowest detection limit of T2 was 10nM. An extended T2E oligonucleotide sequence consisting of T2 sequence and additional 24 nucleotides was shown to cause more pronounced sensor response (at least 5 nM T2E was easily detected). Injection into the sensor cell of the oligonucleotides complementary to the free additional part of T2E after P2-T2E hybridization gave a significant additional SPR response, thus showing that the sandwich hybridization format further improves the sensor sensitivity and decreases the lowest detection limit. The experimental results on surface hybridization between the studied oligonucleotides were in good agreement with thermodynamic parameters of the hybridization calculated for solution conditions. The described approach could be proposed as a basis for creating a biosensor for real-time and label-free diagnostics of drug resistant tuberculosis.

Microbiosensor based on glucose oxidase and hexokinase co-immobilised on platinum microelectrode for selective ATP detection.

ATP determination is of great importance since this compound is involved in a number of vital biological processes. To monitor ATP concentration levels, we have developed a microbiosensor based on cylindrical platinum microelectrode, covered with a layer of poly-m-phenylendiamine (PPD), and layer of co-immobilised glucose oxidase and hexokinase. Conditions for biosensor measurement of ATP (pH, Mg(2+) and substrates concentration) in vitro and microbiosensor characteristics such as sensitivity, selectivity, reproducibility, storage stability were studied and optimized. Under optimal conditions the microbiosensor can measure ATP concentrations down to a 2.5 microM detection limit with response time about 15 s. Interferences by electroactive compounds like biogenic amines and their metabolites, ascorbic acid, uric acid and L-cystein are rejected in general by the PPD layer. The microbiosensor developed is insensitive to ATP analogues (or substances with similar structure), such as ADP, AMP, GTP and UTP, too. It can be used for ATP analysis in vitro in the reactions consuming or producing macroergic triphosphate molecules to study kinetics of the process and in drug design concerning development of inhibitors specific to target kinases and others target enzymes.