ISSN 00036838, Applied Biochemistry and Microbiology, 2013, Vol. 49, No. 3, pp. 306–311. © Pleiades Publishing, Inc., 2013. Original Russian Text © A.G. Burenin, M.P. Nikitin, A.V. Orlov, T.I. Ksenevich, P.I. Nikitin, 2013, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2013, Vol. 49, No. 3, pp. 312–318.
Detection of Pyrethroids by Spectral Correlation Interferometry A. G. Burenina, M. P. Nikitina, A. V. Orlova, T. I. Ksenevichb, and P. I. Nikitinb a
Moscow Institute of Physics and Technology, Moscow, 117303 Russia email:
[email protected] b Prokhorov General Physics Institute, Moscow, 119991 Russia Received September 7, 2012
Abstract—A labelfree method based on spectral correlation interferometry has been developed for highly sensitive detection of pyrethroids by competitive immunoassay on the surface of sensor chips made of widely available microscopy glass cover slips. It is shown that the method allows independent optimization of each step of the sensor surface modification. This fact may be used to increase the efficiency of development of protocols for a wide spectrum of immunoassays that employ glass surface as a solid phase. Detection of 3phenoxybenzoic acid, which is one of the most stable metabolites of a large number of pyrethroids, on the surface of the optimized sensor chips has been demonstrated on the level of 15 pg/ml. That is 50 times better than the sensitivity of the enzymelinked immunosorbent assay (ELISA). DOI: 10.1134/S0003683813030058
INTRODUCTION Pyrethroids are a class of synthetic insecticides dif fering in structure but similar in the mechanism of action to pyrethrins (natural insecticides isolated from plants, such as Pyrethrum cinerariifolium and Tanace tum cinerariifolium). Pyrethroids have been considered safe for humans for a long time [1] which has contrib uted to their wide application in agriculture, veteri nary, and sanitation and epidemiological control. Consequently, these chemicals may enter human body in large amounts together with food and water. The latest studies demonstrated that pyrethroids may neg atively affect the immune system [2], cause disorders of the endocrine system [3], and even possess carcino genic effects [4]. Today, a lot of effort is spent on the development of methods to control these compounds in food, soil, and natural water basins. Since pyrethroid molecules as such degrade rapidly when subjected to light, usually their metabolites are determined. 3Phenoxybenzoic acid (3PBA) is one of the derivatives used most frequently to evaluate the environment and food contamination or the pyre throid dose in the human body. The metabolite was chosen as a marker of pyrethroids due to its stability and universality: it is a product of degradation of a whole series of secondgeneration pyrethroids, for example, cypermethrin, permethrin, and delta methrin [6]. The absence of other abundant sources of Abbreviations: SPR, surface plasmon resonance; DMFA, dimeth ylformamide; 3PBA, 3phenoxybenzoic acid; PBS, phosphate buffered saline; APTES, (3aminopropyl)triethoxysilane; GPTMS, glycidoxypropyltrimethoxysilane.
3PBA makes it the most common marker used in ecological monitoring [7]. To detect molecules as small as 3PBA, liquid chromatography [8] and mass spectrometry [9] are commonly used. These methods are characterized by high sensitivity on the level of dozens of picograms per milliliter. However, the need for timeconsuming sam ple preparation and highcost equipment and con sumables limits their application. Lately, methods of immunological detection of 3PBA have evolved [5, 7]. That is due to the fact that immunoassays substantially reduce the time for sam ple preparation and increase significantly the assay specificity. As a rule, the detection of small molecules is implemented by competitive enzymelinked immu nosorbent assay (ELISA) that allows specific determi nation of a substance through its interaction with anti bodies. The most rapid immunosorbent assays allow one to determine the substance concentration in a sample within 2 h [7] with a detection limit of approx imately several nanograms per milliliter, which is suf ficient for many practical needs. Application of optical labelfree methods based on surface plasmon resonance (SPR) [10] allows one to decrease considerably the assay time, for example, down to 0.5 h in the case of atrazine detection [11]. Moreover, these methods allow the observation of the kinetics of biomolecule adsorption, which increases the assay dynamic range. The commercial SPRbased biosensing systems have proven to be convenient tools for biochemical investigations. However, their employment for ecological monitoring and immuno diagnostics is hampered by the high cost of consum
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ables, i.e., sensor chips. For those detection methods, gold films must be deposited onto the chip surfaces with nanometer precision. Besides, the SPR methods feature parasitic sensitivity to the volume refractive index, which in turn depends strongly on the temper ature of the solution under study. Earlier, the authors proposed a method of spectral correlation interferometry [12, 13] for highly sensitive realtime registration of biochemical reactions on sen sor chips made of affordable microscopy cover slips without any metallic or dielectric coatings. This method allows recording the changes of optical thick ness of a molecular layer adsorbed during biochemical reaction on the sensor chip averaged over the sensing area. The approach provides a signal independent of the solution refractive index. The method of spectral correlation interferometry was successfully applied in various immunochemical studies [14], for elucidation of the mechanisms of drug action [15], in studies of protein interaction with novel polymers [16], and for the detection of a number of autoantibodies in human blood serum [17]. The aim of the work was to adapt the spectral cor relation interferometry method for highly sensitive detection of a number of pyrethroids by their stable metabolite—3PBA—as well as the development of protocols for treatment of glass surface to be used as a solid phase for a wide range of immunoassays. MATERIALS AND METHODS Reagents. In the work, we used sulfuric acid (H2SO4), hydrogen peroxide (H2O2), dimethylforma mide (DMFA) of a special purity grade form Diam (Russia); methanol (СH3OH) pure for analysis from LabScan (United States); biotin, biotin–NHS, bovine serum albumine (BSA), maleimidoRNsuccinimide ester (SigmaAldrich, United States), and streptavidin (Thermo Scientific, United States). All other reagents were of analytical grade. Immunoreagents. 3Phenoxybenzoic acid, 3PBA– BSA conjugate, and rabbit antiserum against the con jugate (1 : 10000) were provided by the State Research Center for Applied Microbiology and Biotechnology (Obolensk, Russia). Buffer preparation. Phosphate buffered saline (PBS) contained 130 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4 (pH 7.4). To prepare a carbonate–bicarbonate buffer (CBB), commercial tablets (pH 8.6; SigmaAldrich) were used. The block ing buffer (BB) contained CBB and 10 mM glycine. The conjugation buffer (CB) contained 100 mM Na2CO3 and 300 mM NaCl (pH 8.5). Spectral correlation interferometry. The progress of biochemical reactions was monitored in real time using an optical labelfree biosensor Picoscope® APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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[16, 17] based on the spectral correlation interferometry. The method allows employment of standard 100μm thick microscopy cover slips as sensor chips. Reagents are supplied using a specially designed microfluidic system with three independent channels, one being a reference one. Two other channels (3 × 2 × 0.1 mm and 0.6 μL each) were used to perform and monitor bio chemical reactions in real time. A control sample, which contained polyclonal antibodies and 3PBA in concentration of 100 ng/mL and produced no signal increment in the used assay format, was passed through one of the the two channels. Experimental samples were passed through the second channel. Immobilization of organosilanes on a glass surface. For the amination and epoxylation of the microscopy cover glass slips, they were incubated in Caro’s acid (30% hydrogen peroxide–sulfuric acid, 1 : 3) for 1 h at 70°C. Then, the slips were bathed in a 5% (3ami nopropyl)triethoxysilane or glycidoxypropyltri methoxysilane solution in methanol. The duration of silanization, as well as the water fraction in the solvent, were optimized for the purposes of the current study. The optimization results are presented below. After three washings with DMFA, the slips were subjected to thermal treatment at 105°C for 1 h. Evaluation of the efficiency of glass silanization. To verify the quality of amination, the glasses were bioti nylated. For this purpose, a mixture of 200 200 μL of DMFA, 12.5 μL of triethylamine, and 1 mg of biotin– NHS was applied to each glass slip. The slips were incubated for 2 h in a chamber that prevented solvent evaporation. After thrice washing with DMFA, the slips were heated to 105°C to remove the physically adsorbed solvent. The modified glass slips were used as the sensor chips. The adsorption of streptavidin on the sensor glass slips from PBS was monitored in the bio sensor microfluidic system at a protein concentration of 50 μg/mL. The quality of slip amination was evalu ated by the response magnitude. To evaluate the efficiency of epoxidation inside the biosensor microfluidic system, adsorption on the sen sor chip of 50 μg/mL immunoglobulin G solution in PBS was monitored. Immobilization of the conjugate onto sensor chips. For aminated chips, maleimidoRNsuccinimide ester was dissolved in DMFA at a concentration of 20 mM and 50 μL of the solution was applied to a sen sor chip. The glass slips were incubated at room tem perature for 3 h. To prevent evaporation, a tightly closed container was used. After three washes with DMFA, 200 μL of 50 μg/mL 3PBA–BSA conjugate in CB was applied to the surface. The slips were incu bated at 4°C for 16 h. The surface was blocked with 3% lowfat milk in PBS during 4 h at room temperature. After three washes with distilled water, the slips were dried and stored at 4°C.
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the signal differed from zero by double the value of the meansquare deviation.
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RESULTS AND DISCUSSION The specificity and sensitivity of immunoassay largely depend on the method of biomolecules immo bilization on a solid phase, since it determines the density of specific adsorption centers and the degree of structure deformation of biorecognition molecules. Therefore, when developing a method to detect small molecules, special attention was paid to the optimiza tion of protocols of conjugate binding to the solid phase. One of the approaches to the immobilization of biomolecules on glass surfaces uses selfassembling monolayers (SAMs) [18], that consists of molecules capable of binding to glass surfaces and to one another. An ordered monolayer formed in this way is covalently bound to a sensor chip at multiple points. Examples of commercially available SAMs include triethoxy and tri methoxysilanes. Aminopropyltriethoxysilane (APTES) and glycidoxypropyltrimethoxysilane (GPTMS) are convenient to use for protein assays. Upon immobiliza tion of these substances on a glass surface, an ordered layer of amino or epoxy groups is formed which may be further used for the covalent binding of biomolecules or lowmolecularweight ligands. Due to the multistep nature of protein immobiliza tion on sensor chips, the development of the relevant protocols by traditional methods, which use fluores cent or enzyme labels, is compicated. These methods allow one to observe the final result only and require the simultaneous optimization of all assay stages. The spectral correlation interferometry simplifies and con siderably accelerates the process of protocol develop ment, since it permits realtime monitoring of all immunoassay stages. Therefore, optimization of each step may be performed independently of all others. The first stage of immobilization of biomolecules onto a microscopy glass cover slip employed in the proposed method as a sensor chip is modification of its surface with organosilanes. To optimize the step, we studied the dependence of the adsorption center den sity on the modified glass slips upon silanization dura tion and water content in the solution. To evaluate the adsorption center density with the Picoscope® bio sensor, we monitored the specific protein adsorption as described in the Materials and Methods section. The signal obtained for the aminated glass slips as a function of incubation time is presented in Fig. 1. As follows from the figure, the maximum adsorp tion capacity, which differed from the maximal value obtained in the experiments by less than 5%, was reached after 16 h. This incubation time is optimal for practical use, because any further increase of the incu
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Fig. 1. Signal (nm) observed upon streptavidin adsorption as a function of amination time (h).
Immobilization of the conjugate on the surface of epoxylated sensor chips was performed immediately before the assay in the biosensor flow system using CBB (pH 8.6) at a conjugate concentration of 50 μg/mL. To decrease nonspecific binding, the glass surface was additionally treated with BB. Preparation of test samples. 3PBA was dissolved in a water–methanol mixture (6 : 4) at the necessary con centration. Then, BSA was added at a concentration of 50 μg/mL. Prior to the assay, 20 μL antiserum per milliliter of sample was added to the solution. Competitive immunoassay. In the microfluidic sys tem of the biosensor, the adsorption of free polyclonal antibodies from the sample onto the sensor chip with immobilized conjugate was monitored. The solution flow rate was set at 5 μL/min. A change in the biolayer thickness registered when the sample passed over the slip was considered to be an analytical signal. To con trol the value of nonspecific signals, two types of experiments were performed: either sensor chips with immobilized BSA containing no sites for specific adsorption or samples with 3PBA in concentration of 100 ng/mL (enough to bind all antibodies in the sam ple) were used. Statistical data processing. In each series of exper iments, the resulting value was calculated as the arith metic mean of the measurements of separate experi ments in this series while the error, as the meansquare deviation. The detection limit was determined accord ing to the two σ criterion, that is, it was equal to the concentration under which the signal differed from the one obtained in the absence of the antigen by dou ble the value of the meansquare deviation of the blank control. The value of the upper limit of the dynamic range was calculated as the concentration under which
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bation duration caused degradation of the optical quality of the sensor chips due to the adsorption of polymerized agglomerates of APTES. Only slight dependence of amination efficiency on water content was experimentally observed while the parameter varied in the range of 0.01–3%. In further experiments, the glass slips were aminated during 16 h at water content in the methanol solution 1%. The maximum value of the signal upon streptavidin adsorption was 1.2 nm. According to a theoretical esti mate, immobilization of a dense monolayer of the pro tein should produce a signal equivalent to approxi mately 3.5 nm. Therefore, the developed amination protocol allows one to cover 30% of the chip surface. In the process of optimization of epoxidation pro tocols for the glass slips, we showed experimentally that the signal magnitude upon protein adsorption on the sensor chips does not depend on silanization time within 2–24 h. The dependence on the water content in solution is more complicated and reaches its maxi mum at 0.1%. According to the experimental data, epoxidation time of 16 h and water content of 0.1% are optimal for the sensor chip production. Streptavidin adsoprtion onto the glass chips prepared according to this protocol resulted in a signal of 2.4 nm, which cor responds to 60% coverage of thesensor chip surface. The conducted experiments resulted in protocols for fabrication of amino and epoxy sensor chips. The latter are characterized by a twofold higher adsorption capacity, which is equivalent to the increased number of immobilized centers of specific adsorption. The epoxy chips also allow employment of simple and con venient protocols of protein immobilization through covalent binding with protein amino groups. As for many practical needs the shelf life of sensor chips is an important factor; a series of experiments was per formed to study the dependence of the registered sig nal on the storage time of the modified glass. It was found that with the developed protocols of epoxida tion the signal stays unchanged during storage for up to half a year. For example, the difference between sig nals observed upon protein adsorption onto an epoxy chip immediately after its preparation and after stor age during 6 months at + 4°C did not exceed 10%. Since a major reason for reduction of the signal speci ficity is the hydrolysis of surface epoxy groups, storage in an inert atmosphere may additionally increase their shelf life. The slide amination reduces the effect of storage duration: the surface provides a stable signal for over 1 year. However, immobilization of biomole cules onto the aminated surface requires more sophis ticated protocols, including preliminary activation that further increases the assay time. Since there are practical tasks demanding advanta geous properties for both investigated types of the sen sor chips, further experiments were performed with APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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Fig. 2. Sensograms obtained upon the successive pumping along an epoxy sensor chip in the microfluidic system of the Picoscope® biosensor of the following substances: I, 3 PBA–BSA conjugate; II, carbonate–bicarbonate buffer sup plemented with glycine; and III, experimental sample. 1, blank control (a sample containing no 3PBA); 2, 3PBA sample at a concentration of 100 ng/mL.
both amino and epoxy sensor chips prepared accord ing to the optimized protocols. The developed sensor chips were used for compet itive immunoassay to detect lowmolecularweight molecules. Adsorption of polyclonal antibodies from samples prepared as described in the Materials and Methods section, which contained different concen trations of 3PBA, onto a sensor chip surface with immobilized 3PBA–BSA conjugate, was observed in real time by the Picoscope® biosensor. A typical sen sogram is presented in Fig. 2. An increase in the biolayer thickness during pump ing of the sample is considered as signal Δ. As follows from Fig. 2, specific adsorption of antibodies from sam ples with high concentrations of 3PBA was absent. Similarly, in the control experiments, no signal was observed when the samples were pumped along the sur face with immobilized BSA. This indicated the absence of nonspecific binding of the sample components to the surface. In the absence of 3PBA in the samples pre pared for aminated glass slips, the signal value was 0.3 nm with a meansquare deviation of 0.02 nm; for epoxy ones, it was 1.1 and 0.05 nm, respectively. The signal dependence on the 3PBA concentra tion upon antibody adsorption from a sample prepared for epoxy slides is presented in Fig. 3a. As follows from the figure, the limit of 3PBA detection on the epoxy chips was 0.2 ng/mL. That is by almost an order of magnitude better than for the existing ELISA methods of pyrethroid metabolite determination, which allow for the detection of concentrations above 1 ng/mL [7]. The dynamic range of the assay consists of three orders of magnitude which encompasses the whole range of
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Fig. 3. Signal (nm) observed upon competitive immunoanalysis as a function of the 3PBA concentration (ng/mL): (a) epoxy chips and (b) amino chips.
concentrations important from the point of view of ecological monitoring and toxicological analysis. Further improvements in the limit of detection were achieved by immobilization of the conjugate onto the surface of aminated sensor chips. The signal dependence on the 3PBA concentration in a sample is presented in Fig. 3b. As follows from the figure, the 3PBA detection limit on aminated slips was 0.015 ng/mL. That is an order of magnitude better than the results obtained with the epoxy chips and is at the same level as the results produced by the methods of liquid chromatography and mass spectrometry [19]. This was achieved owing to the low variance of the results and the high slope of the curve close to the detection limit values. The dynamic range is two orders of magnitude. The wider dynamic range for epoxy chips may be explained by their higher adsorp tion capacity. The detection limits achieved in both cases permit employment of the epoxy and amino sensor chips for the purposes of highsensitive biosensorics. A wide dynamic range, convenience of work, and timesaving assay make the epoxy chips an attractive solution for the toxicological screening of food products. The high sensitivity obtained in the case of aminated chips may be of demand in ecological monitoring and control of potentially dangerous production units. Thus, the protocols for modification of the sensor chip surface were optimized, and the developed method of optical monitoring of competitive immu nochemical reactions was adopted to allow rapid highly sensitive determination of lowmolecular weight molecules with high sensitivity. The efficiency of the approach was demonstrated by the detection of 3PBA. A detection limit of 15 pg/mL was reached. That surpasses the existing methods of pyrethroid detection by immunoassay by a factor of over 50 and is
at the same level as modern methods of liquid chroma tography and mass spectrometry. The use of standard microscopy glass cover slips as widely available con sumables—sensor chips, as well as the compactness and simplicity of the used equipment, together with low time consumption, allow one to apply the tech nology not only in research but for ecological moni toring. ACKNOWLEDGMENTS We are grateful to the State Research Center of Applied Microbiology (Obolensk, Russia) for provid ing immunoreagents. The work was partially supported by the Ministry of Education and Sciences of the Russian Federation (projects nos. 16.512.11.2124 and 14.740.11.1179) and the Russian Foundation for Basic Research (project nos. 110412181, 100201185, and 1102 01440). REFERENCES 1. Class, T.J. and Kintrup, J., J. Anal. Chem., 1991, vol. 340, no. 7, pp. 446–453. 2. Hadnagy, W., Leng, G., Sugiri, D., Ranft, U., and Idel, H., Int. J. Hygiene Environ. Health, 2003, vol. 206, no. 2, pp. 93–102. 3. Liu, J., Yang, Y., Zhuang, S., Yang, Y., Li, F., and Liu, W., Toxicology, 2011, vol. 290, no. 1, pp. 42–49. 4. Fortes, C., Cancer, in Encyclopedia of Environmental Health, New York: Elsevier, 2011, pp. 489–497. 5. Zherdev, A.V., Dzantiev, B.B., and Trubaceva, J.N., Anal. Chim. Acta, 1997, vol. 347, nos. 1–2, p. 13. 6. Kaneko, H., J. Agric. Food Chem., 2011, vol. 59, no. 7, pp. 2786–2791. 7. Wang, J., Yu, G., Sheng, W., Shi, M., Guo, B., and Wang, S., J. Agric. Food Chem., 2011, vol. 59, no. 7, pp. 2997–3003.
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