Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor

We constructed a biosensor by electrodeposition of gold nano-particles (AuNPs) on glassy carbon (GC) and subsequent formation of a 4-mercaptobenzoic acid self-assembled monolayer (SAM). The enzyme horseradish peroxidase (HRP) was then covalently immobilized onto the SAM. Two forms of HRP were employed: non-modified and chemically glycosylated with lactose. Circular dichroism (CD) spectra showed that chemical glycosylation did neither change the tertiary structure of HRP nor the heme environment. The highest sensitivity of the biosensor to hydroquinone was obtained for the biosensor with HRP-lactose (414 nA μM⁻¹) compared to 378 nA μM⁻¹ for the one employing non-modified HRP. The chemically glycosylated form of the enzyme catalyzed the reduction of hydroquinone more rapidly than the native form of the enzyme. The sensor employing lactose-modified HRP also had a lower limit of detection (74 μM) than the HRP biosensor (83 μM). However, most importantly, chemically glycosylation improved the long-term stability of the biosensor, which retained 60% of its activity over a four-month storage period compared to only 10% for HRP. These results highlight improvements by an innovative stabilization method when compared to previously reported enzyme-based biosensors.     

Acrylamide concentrations in grilled foodstuffs of Turkish kitchen by high performance liquid chromatography-mass spectrometry

For over ten years, there has been a considerable interest in determination of acrylamide in foodstuffs. It was known that both protein-rich and carbohydrates-rich foods cooked at high-temperatures can cause acrylamide formation. However, carbohydrates-rich foods such as potato chips and biscuit samples have been the common studied foods compared with protein-rich foods such as meat samples.In this study, determination of acrylamide in these two group foods was examined using HPLC-MS. For this purpose, firstly, the parameters that are thought to affect the response in the HPLC-MS analysis were optimized. The optimized conditions were found to be 0.3 ml min^{− 1} for flow rate of mobile phase, 40 µl for injection volume, 5 °C for column temperature and 70 V for fragmentor potential. The optimized method was applied for the determination of acrylamide levels in Turkish foodstuffs including grilled meat and chicken samples, potato chips, coffee and biscuit. The obtained concentrations for all studied foods were in the range of 20–250 µg kg^{− 1}. The results showed that acrylamide concentrations highly varied depending on the kind of food samples.     

Simultaneous determination of 3-monochloropropane-1,2-diol and acrylamide in food by gas chromatography-triple quadrupole mass spectrometry with coupled column separation

Both 3-monochloropropane-1,2-diol (3-MCPD) and acrylamide are contaminants found in heat-processed foods and their related products. A quantitative method was developed for the simultaneous determination of both contaminants in food by gas chromatography-triple quadrupole mass spectrometry (GC–MS/MS). The analytes were purified and extracted by the matrix solid-phase dispersion extraction (MSPDE) technique with Extrelut NT. A coupled column (a 3 m Innowax combined with a 30 m DB-5 ms) was developed to separate both compounds efficiently without derivatization. Triple quadrupole mass spectrometry in multiple reaction monitoring mode (MRM) was applied to suppress matrix interference and obtain good sensitivity in the determination of both analytes. The limit of detection (LOD) in the sample matrix was 5 μg kg⁻¹ for 3-MCPD or acrylamide. The average recoveries for 3-MCPD and acrylamide in different food matrices were 90.5–107% and 81.9–95.7%, respectively, with the intraday relative standard deviations (RSDs) of 5.6–13.5% and 5.3–13.4%, respectively. The interday RSDs were 6.1–12.6% for 3-MCPD and were 5.0–12.8% for acrylamide. Both contaminants were found in samples of bread, fried chips, fried instant noodles, soy sauce, and instant noodle flavoring. Neither 3-MCPD nor acrylamide was detected in the samples of dairy products (solid or liquid samples) and non-fried instant noodles.     

Gold nanoparticle–antibody conjugates for specific extraction and subsequent analysis by liquid chromatography–tandem mass spectrometry of malondialdehyde-modified low density lipoprotein as biomarker for cardiovascular risk

Oxidized low-density lipoproteins (OxLDLs) like malondialdehyde-modified low-density lipoprotein (MDA-LDL) play a major role in atherosclerosis and have been proposed as useful biomarkers for oxidative stress. In this study, gold-nanoparticles (GNPs) were functionalized via distinct chemistries with anti-MDA-LDL antibodies (Abs) for selective recognition and capture of MDA-LDL from biological matrices. The study focused on optimization of binding affinities and saturation capacities of the antiMDA-LDL-Ab-GNP bioconjugate by exploring distinct random and oriented immobilization approaches, such as (i) direct adsorptive attachment of Abs on the GNP surface, (ii) covalent bonding by amide coupling of Abs to carboxy-terminated-pegylated GNPs, (iii) oriented immobilization via oxidized carbohydrate moiety of the Ab on hydrazide-derivatized GNPs and (iv) cysteine-tagged protein A (cProtA)-bonded GNPs. Depending on immobilization chemistry, up to 3 antibodies per GNP could be immobilized as determined by ELISA. The highest binding capacity was achieved with the GNP-cProtA-Ab bioconjugate which yielded a saturation capacity of 2.24 ± 0.04 μg mL⁻¹ GNP suspension for MDA-LDL with an affinity K_d of 5.25 ± 0.11 × 10⁻¹⁰ M. The GNP-cProtA-antiMDA-LDL bioconjugate revealed high specificity for MDA-LDL over copper(II)-oxidized LDL as well as native human LDL. This clearly demonstrates the usefulness of the new GNP-Ab bioconjugates for specific extraction of MDA-LDL from plasma samples as biomarkers of oxidative stress. Their combination as specific immunoextraction nanomaterials with analysis by LC–MS/MS allows sensitive and selective detection of MDA-LDL in complex samples.     

Optimisation of a liquid chromatography-tandem mass spectrometric method for the determination of acrylamide in foods

A robust and fitted routine method resulting from an analytical optimisation has been applied for the determination of acrylamide in several foods including mainly potato and cereal products. For the sample treatment, different materials were evaluated for filtration and purification of the extract. To increase the performances in terms of sensitivity, a preconcentration to small volume was introduced before liquid chromatography coupled to tandem mass spectrometry analysis on a μ-Bondapak C₁₈ column using d₃-acrylamide as internal standard. For identification, relative retention time and two diagnostic ions were monitored. A limit of detection of 10 μg kg⁻¹, a limit of quantitation of 20 μg kg⁻¹, mean recoveries ranging from 100 to 115%, coefficients of variation from 1.36 to 8.06% for repeatability and from 3.3 to 18.2% for reproducibility within the laboratory and a measurement uncertainty of 42% were obtained during an in-house validation procedure. Results of tests, validation data and Z-score obtained during participation to proficiency studies are presented.     

Determination of phosphoamino acids by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

A micellar electrokinetic capillary chromatography (MEKC) method with laser-induced fluorescence detection (LIF) was developed for analyzing three phosphoamino acids including phosphotyrosine (P-Tyr), phosphoserine (P-Ser), and phosphothreonine (P-Thr). 3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for derivatization of these phosphoamino acids. Results indicated that the complete baseline resolution of each phosphoamino acid was obtained within 10 min, using 20 mmol l⁻¹ sodium borate buffer (pH 9.35) containing 20 mmol l⁻¹sodium deoxycholate (SDC) and 10 mmol l⁻¹ Brij35. Other common amino acids, especially Glu and Asp, did not disturb the assay of these phosphoamino acids. There was a linear relationship between the peak area for analyte and its concentration, with correlation coefficients in the range of 0.9966-0.9996. The concentration detection limits (signal-to-noise = 3) for P-Tyr, P-Ser, and P-Thr were 10, 40, and 75 nmol l⁻¹, respectively. The developed method was successfully applied for determining phosphoamino acids in the hydrolysis sample of a phosphorylated protein kinase.     

Determination of acrylamide in food by solid-phase microextraction coupled to gas chromatography-positive chemical ionization tandem mass spectrometry

A method has been developed to determine acrylamide in aqueous matrices by using direct immersion solid-phase microextraction (SPME) coupled to gas chromatography-positive chemical ionization tandem mass spectrometry (GC-PCI-MS-MS) in the selected reaction monitoring (SRM) mode. The optimized SPME experimental procedures to extract acrylamide in water solutions were: use of a carbowax/divinylbenzene (CW/DVB)-coated fiber at pH 7, extraction time of 20 min and analyte desorption at 210 °C for 3 min. A detection limit of 0.1 μg L⁻¹ was obtained. The linear range was 1-1000 μg L⁻¹. The relative standard deviation was 10.64% (n = 7). The proposed analytical method was successfully used for the quantification of trace acrylamide in foodstuffs such as French fries (1.2 μg g⁻¹) and potato crisps (2.2 μg g⁻¹).     

Piezoelectric affinity sensors for cocaine and cholinesterase inhibitors

We report here the development of piezoelectric affinity sensors for cocaine and cholinesterase inhibitors based on the formation of affinity complexes between an immobilized cocaine derivative and an anti-cocaine antibody or cholinesterase. For both binding reactions benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on the surface of the sensor. For immobilization, pre-conjugated BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU) allowed the formation of a chemisorbed monolayer on the piezosensor surface.The detection of cocaine was based on a competitive assay. The change of frequency measured after 300 s of the binding reaction was used as the signal. The maximum binding of the antibody resulted in a frequency decrease of 35 Hz (with an imprecision 3%, n = 3) while the presence of 100 pmol l⁻¹ cocaine decreased the binding by 11%. The limit of detection was consequently below 100 pmol l⁻¹ for cocaine. The total time of one analysis was 15 min.This BZE-DADOO-modified sensor was adapted for the detection of organophosphates. BZE-DADOO - a competitive inhibitor - served as binding element for cholinesterase in a competitive assay.     

Electropolymerized surface ion imprinting films on a gold nanoparticles/single-wall carbon nanotube nanohybrids modified glassy carbon electrode for electrochemical detection of trace mercury(II) in water

Electrochemical detection of Hg(II) using a electropolymerized ion imprinting poly(2-mercaptobenzothiazole) films at the surface of gold nanoparticles/single-walled carbon nanotube nanohybrids modified glassy carbon electrode (PMBT/AuNPs/SWCNTs/GCE) is described for the first time. The Hg(II)-imprinted PMBT/AuNPs/SWCNTs/GCE sensor exhibits larger binding to functionalized capacity, larger affinity, faster binding kinetics and higher selectivity to template Hg(II). The differential pulse anodic stripping voltammetry (DPASV) response of the Hg(II)-imprinted PMBT/AuNPs/SWCNTs/GCE sensor to Hg(II) is ca. 3.7- and 10.5-fold higher than that at the non-imprinted PMBT/AuNPs/SWCNTs/GCE and the imprinted PMBT/AuNPs/GCE, respectively, and the detection limit for Hg(II) is 0.08 nM (S/N = 3, which is well below the guideline value given by the World Health Organization) and a sensitivity of 0.749 μA nM⁻¹ was obtained. Excellent wide linear range (0.4–96.0 nM) and good repeatability (relative standard deviation of 2.6%) were obtained for Hg(II). The interference experiments show that Ag(I), Pb(II), Cd(II), Zn(II) and Cu(II) had little or no influence on the Hg(II) signal. These values, particularly the high sensitivity and excellent selectivity in contrast to the values reported previously in the area of electrochemical Hg(II) detection, demonstrate the analytical performance of the Hg(II)-imprinted PMBT/AuNPs/SWCNTs/GCE toward Hg(II) is superior to the existing electrodes and could be used for efficient determination of Hg(II) in natural water samples.     

Development of an immunoassay and a sol-gel-based immunoaffinity cleanup method for coplanar polychlorinated biphenyls from soil and sediment samples

Two polychlorinated biphenyls (PCB) enzyme linked immunosorbent assays (ELISAs) were developed using goat PCB purified immunoglobulin (IgG) antibodies (Abs). The IgGs exhibited the highest affinity toward PCB-77 (24 ng mL⁻¹) with sensitivities in the range of 6-11 ng mL⁻¹. The Abs cross-reacted with PCB-126 and the heptachlorodibenzofuran 1,2,3,4,6,7,8-HpCDF but not with PCB-169, PCB-118, Aroclor 1232, 1248, 1260 or the hexachlorodibenzofuran 2,3,4,6,7,8-HxCDF. The IgGs were also used to develop a sol-gel-based immunoaffinity purification (IAP) method for cleanup of PCB-126. Recovery efficiencies depended on the sol-gel formats; a 1:12 format resulted in the highest binding capacity. Net binding capacity ranged from 112 to 257 ng, and 90% of the analyte could be eluted with only 2 mL of ethanol. The method was also very efficient in purifying PCB-126 from spiked soil and sediment samples from contaminated sites; and eliminating matrix interferences to a degree that enabled analysis of the purified samples by ELISA. The approaches developed in the course of the study form a basis for the development of additional IAP methods for other PCBs, and their implementation in high-throughput screening programs for PCB in food, soil, and other environmental and biological samples.     

A dendrimer-based immunosensor for improved capture and detection of tumor necrosis factor-α cytokine

A dendrimer-based sandwich type enzyme-linked immunosorbent assay (ELISA) was developed for the improved detection of recombinant human tumor necrosis factor-alpha (TNF-α) for early diagnosis of perinatal diseases. Hydroxyl-terminated generation four poly(amidoamine) dendrimer (G4-OH) was used for the development of a solid phase bio-sensing platform. The surface of the ELISA plate was modified with polyethylene-glycol (PEG) and thiol-functionalized G4-OH was immobilized on the PEG-functionalized plate. A capture antibody was oxidized and covalently immobilized onto the dendrimer-modified ELISA plate, which provides favorable orientation for the antigen binding sites toward the analyte. The dendrimer-modified plate showed enhanced sensitivity, and the detection limit for TNF-α was found to be 0.48 pg mL⁻¹, which is significantly better than the commercially available ELISA kit. The selectivity of the dendrimer-modified ELISA plate was further evaluated with a mixture of cytokines, which showed results for similar to that of TNF-α alone. The modified plate provides a greater opportunity for the detection of a wide range of cytokines and biomarkers.     

平面金基质上硅壳的构筑

成功地在平板金基质上构筑了硅壳. 在构筑硅壳前, 首先通过形成Au-S键将具有双官能团的4-巯基苯甲酸自组装到金膜上, 再通过静电吸附, 将3-氨丙基-三乙氧基硅烷偶连到4-巯基苯甲酸的末端羧基上, 然后通过碱催化水解3-氨丙基-三乙氧基硅烷和四乙氧基硅烷, 形成交联的Si-O-Si键, 构筑成硅壳. 利用原子力和扫描电子显微镜表征了硅壳的形貌, 利用红外和X射线光电能谱研究了它的结构. 此硅壳易于衍生成不同官能团的探针, 可进一步用来构建分子传感和识别单元.     

Separation and determination of acrylamide in potato chips by micellar electrokinetic capillary chromatography

A simple and rapid method using micellar electrokinetic capillary chromatography (MEKC) was developed for the separation and determination of acrylamide in potato chips at low levels for the first time. The experimental conditions for the separation and quantification of acrylamide were optimized at first. The optimized conditions were: 50 mmol/L Na₂B₄O₇ and 40 mmol/L SDS at pH 10.0, 12 kV applied voltage, 76 cm total length (67 cm effective length) and 75 μm i.d. capillary, 198 nm wavelength, 15 cm high 25 s hydrodynamics sample injection, 20 °C air-cooling. The linear response of acrylamide concentration ranges from 0.5 to 100 μg/mL with high correlation coefficient (r = 0.9986, n = 9). The LOD and LOQ were estimated to be 0.1 and 0.33 μg/mL based on S/N = 3 and 10. The precision values (expressed as R.S.D.) of intra- and inter-day were 0.86-4.35% and 2.61-9.65%, respectively. Recoveries spiked at levels 2, 20, 60 μg/mL ranged between 90.86% and 99.6% with R.S.D. less than 6.5%. Finally, the developed method has been applied to the analysis of real samples and has achieved satisfactory results. All of these indicated that it was a reliable method for the quantification of acrylamide in potato chips.     

Monitoring of progestins: Development of immunochemical methods for purification and detection of levonorgestrel

A polyclonal antibody (Ab) for the progestin levonorgestrel (LNG) was generated, and immunochemical assays for its detection, clean-up and concentration were developed. A highly specific microplate diagnostic assay for the detection of LNG was developed that used the enzyme linked immunosorbent assay (ELISA) method. The LNG ELISA developed was sensitive and reproducible; it exhibited I₅₀ and I₂₀ values of 3.3 ± 1.8 ng mL⁻¹ and 0.6 ± 0.4 ng mL⁻¹, respectively, and the Abs did not cross react with any of the tested steroid hormones. The above Abs were used to develop a sol-gel-based immunoaffinity purification (IAP) method for concentration and clean-up of LNG that is compatible with subsequent immunochemical or instrumental chemical analytical procedures, such as liquid chromatography followed by mass spectrometry (LC-MS/MS). Development of the columns included successful entrapment of Abs within a tetramethoxysilane (TMOS)-based SiO₂ polymer network. The Abs could bind the free analyte from solution, and the bound analyte could be easily eluted from the sol-gel matrix at high recoveries. The Ab selectivity towards the antigen was high, in both ELISA and the sol-gel columns, but the entrapped Abs cross-reacted with two other steroid hormones - ethynylestradiol (EE2) and nortestosterone (NT) - which share similar epitopes with LNG, despite the lack of cross reactivity in the ELISA. The validity of the method was investigated by LC-MS/MS and a good analytical correlation was obtained.     

Ultrasound wave-mediated enzyme-linked immunosorbent assay technique

With the increasing burden of infectious diseases, it has become important to develop a rapid ELISA that could facilitate early diagnosis. Herein, we have shown that ultrasound waves can dramatically reduce the ELISA timing without losing its specificity or sensitivity. Ultrasound-mediated ELISA was best achieved on an activated microtiter plate which was able to covalently bind antigen or antibody in 10 min when subjected to ultrasound waves in a sonicator bath having a temperature of 37 °C, operating at a frequency of 40 KHz and an output power of 120 W. Blocking, antibody binding and secondary antibody-enzyme conjugate binding were also accomplished in 10 min each in the sonicator bath under similar conditions. The validation of SELISA method was demonstrated by detecting IgE in allergic patient's sera. Total IgE detection by 40 min-SELISA method gives similar absorbance value to that obtained by 20 h conventional or 3 h-HELISA procedure. As SELISA method can detect IgE even at the serum dilution of 1/50 (v/v) on photoactivated surface it could be significantly useful to confirm false negative cases. The inhibition assay ruled out the possibility of any cross reactivity or non-specific binding. As SELISA procedure is sensitive, specific and reproducible (intra- and inter-assay CVs were 9.65% and 8.47%) it could be an excellent alternative to conventional ELISA or HELISA procedures.     

A generic method for the determination of acrylamide in thermally processed foods

A generic sample preparation method for the determination of acrylamide in foods was developed. The method entails extraction with methanol, purification with Carrez I and II solutions, evaporation and solvent change to water, and cleanup with Oasis HLB solid-phase extraction (SPE) cartridge. The final extract was analyzed by liquid chromatography-mass spectrometry (LC-MS) for quantitation. The chromatographic separation was performed on ODS-3 column using the isocratic mixture of 0.01 mM acetic acid in 0.2% aqueous solution of formic acid at a flow rate of 0.6 ml/min at 25 degrees C. The recoveries of acrylamide from potato chips, biscuits and coffee ranged between 92.8 and 101.5% with relative standard deviations of 4.1% or less. The limit of detection (LOD) and the limit of quantitation (LOQ) were 2 ng/g and 6 ng/g in the basis of signal to noise ratios of 3:1 and 9:1, respectively.     

An efficient synthesis of 3-(indol-3-yl)quinoxalin-2-ones with TfOH-catalyzed Friedel-Crafts type coupling reaction in air

An efficient one-pot synthetic approach to access a variety of 3-(indol-3-yl)quinoxalin-2-ones from various quinoxalin-2-ones and very wide scope of indoles through TfOH-catalyzed Friedel-Crafts type coupling reaction in DMF has been developed. Only 10 mol % Brønsted acid as a catalyst, air as an oxidizer, and very wide range of substrates are the prominent advantages of this method.     

Solvent effects on the copolymerization kinetics of ionic (AMPS) and non-ionic (HEAm) acrylamide derivatives

The free-radical copolymerization of two N-substituted acrylamide monomers, the ionic AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid) and the non-ionic HEAm (2-hydroxyethylacrylamide) is presented. Despite bearing similar polymerizable functionalities, HEAm is more reactive toward free-radical addition than AMPS in water. In a mixed aqueous solvent containing salt, (0.5 M LiNO₃, 50 wt%) and ethanol (50 wt%), the reactivity ratio was found to be r_AMPS = 0.53 and r_HEAm = 1.06 indicating that copolymers with a nearly random distribution of sulfonic and hydroxy functionalities can be prepared.     

Determination of acrylamide in Chinese traditional carbohydrate-rich foods using gas chromatography with micro-electron capture detector and isotope dilution liquid chromatography combined with electrospray ionization tandem mass spectrometry

The present study developed two analytical methods for quantification of acrylamide in complex food matrixes, such as Chinese traditional carbohydrate-rich foods. One is based on derivatization with potassium bromate and potassium bromide without clean-up prior to gas chromatography with micro-electron capture detector (GC-MECD). Alternatively, the underivatized acrylamide was detected by high-performance liquid chromatography coupled to quadrupole tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization mode. For both methods, the Chinese carbohydrate-rich samples were homogenized, defatted with petroleum ether and extracted with aqueous solution of sodium chloride. Recovery rates for acrylamide from spiked Chinese style foods with the spiking level of 50, 500 and 1000 μg kg⁻¹ were in the range of 79-93% for the GC-MECD including derivatization and 84-97% for the HPLC-MS/MS method. Typical quantification limits of the HPLC-MSMS method were 4 μg kg⁻¹ for acrylamide. The GC-MECD method achieved quantification limits of 10 μg kg⁻¹ in Chinese style foods. Thirty-eight Chinese traditional foods purchased from different manufacturers were analyzed and compared with four Western style foods. Acrylamide contaminant was found in all of samples at the concentration up to 771.1 and 734.5 μg kg⁻¹ detected by the GC and HPLC method, respectively. The concentrations determined with the two different quantitative methods corresponded well with each other. A convenient and fast pretreatment procedure will be optimized in order to satisfy further investigation of hundreds of samples.     

Development of an indirect competitive ELISA for the determination of papaverine

Papaverine (1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, PAP) is a member of the benzylisoquinoline sub-group of the opium alkaloids. It has been widely used for treating diseases like pulmonary arterial embolism and renal or biliary colic. In this paper, a specific conjugate of mono-demethylated papaverine-O-carboxylmethyl ether (MDMPAP-O-CME) and bovine serum albumin (BSA) was synthesized and used as the complete antigen (PAP-BSA), with which we successfully obtained a high-titer anti-PAP polyclonal antibody (pAb) by immunization of rabbits. The anti-PAP pAb showed high affinity to papaverine with an affinity constant (K_aff) of 7.3 × 10⁷ L/mol. With this antibody, we established a sensitive immunochemical method for the determination of papaverine based on indirect competitive enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of the coated antigen (PAP-OVA) and purified pAb used in the ELISA were 5 and 1.2 μg/mL, respectively. The cross reactivity of other benzylisoquinoline derived substances, including 1-(3,4-dihydroxybenzyl)-7-hydroxy-6-methoxy-isoquinoline (6-methoxy-papaveroline, MPAPO), morphine (MP) and codeine (CD) were all lower than 1%. The linear range of the calibration curve was 0.1-1000 ng/mL. Normal human serum samples were spiked with known amount of papaverine and measured by the ELISA. Recoveries were between 102% and 105%. Papaverine content in a commercial papaverine hydrochloride injection sample was also determined using the established ELISA. Compared with the results given by the control experiment of HPLC, the recoveries of ELISA to detect injection samples were 102-110%. The limits of detection for synthetic serum samples and injection samples of papaverine hydrochloride were 0.25 and 0.06 ng/mL, respectively.