Spectrophotometric-Dual-Enzyme-Simultaneous Assay in One

Jan 10, 2013 - College of Laboratory Medicine, Chongqing Medical University, ... For serum enzyme assays in laboratory medicine, high-throughput scree...
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Spectrophotometric-Dual-Enzyme-Simultaneous Assay in One Reaction Solution: Chemometrics and Experimental Models Hongbo Liu,¶ Xiaolan Yang,¶ Lin Liu, Jizheng Dang, Yanling Xie, Yi Zhang, Jun Pu, Gaobo Long, Yuanli Li, Yonghua Yuan, Juan Liao, and Fei Liao* Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China S Supporting Information *

ABSTRACT: Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (λA) much larger than that of Product B (λB) formed by Enzyme B action on Substrate B; λB is close to the longest isoabsorbance wavelength of Product A and Substrate A (λ0); (b) absorbance at λA and λ0 is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with γ-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. γ-Glutamyltransferase releases p-nitroaniline from γ-glutamyl-p-nitroaniline with λ0 at 344 nm and λA close to 405 nm, lactatedehydrogenase consumes reduced nicotinamide dinucleotide bearing λB at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-nitroaniline; the linear range of initial rates of γ-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and β-D-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with λ0 at 405 nm and λA at 458 nm, β-D-galactosidase releases 4-nitrophenol from β-D(4-nitrophenyl)-galactoside with λB at 405 nm. No interference from substrates/products made SDESA of β-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising.

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inhibition/activation of each enzyme by all involved substances and to have favorable limits, sensitivity, and linear ranges for quantifying all enzymes. Hence, simultaneous assay of two enzymes in one reaction solution is investigated in this report. Spectrophotometric methods with chromogenic substrates are the most favorable for enzyme assay. Currently, conventional spectrophotometers and microplate readers already can quantify the absorbance of one solution simultaneously at multiple wavelengths via swift alteration of detection wavelengths. By concurrently initiating the reactions of two enzymes on their chromogenic substrates, spectrophotometric-dual-enzymesimultaneous assay in one reaction solution (SDESA) may be fulfilled by continuously recording the absorbance of the reaction solution concomitantly at two wavelengths for two chromogenic substrates/products. Inevitably, SDESA should stand with the potential negative impacts of the same buffer and pH

or serum enzyme assays in laboratory medicine, high-throughput screening of enzyme inhibitors, and enzyme-linkedimmunoabsorbent assay (ELISA), activities of some enzymes are measured repetitively. Initial rate and maximum reaction rate (Vm) indexing enzyme activities can be interconverted according to known kinetics. The classical initial rate method is the commonest to determine enzyme initial rate from data of linear drop of substrate levels or initial rate reaction.1,2 Other types of methods estimate Vm or initial rate via kinetic analysis of reaction curve with data beyond initial rate reaction.3−6 However, all those methods measure only one enzyme of interest in one reaction solution and display limited efficiency. To enhance analysis efficiency, an absorbing strategy is simultaneous assay of multiple enzymes in one reaction solution by concomitantly initiating their reactions and concurrently quantifying their products/substrates. This strategy has been proposed for ELISA, but no reports of this strategy initiate the reactions of multiple enzymes as ELISA labels and quantify their substrates/ products concomitantly in the same reaction solutions.7−14 The simultaneous assay of more enzymes in one reaction solution exhibits better efficiency, but it is tougher to process the potential © 2013 American Chemical Society

Received: September 25, 2012 Accepted: January 10, 2013 Published: January 10, 2013 2143

dx.doi.org/10.1021/ac302786p | Anal. Chem. 2013, 85, 2143−2154

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Mining the Interference-Free Absorbance of Two Chromogenic Products/Substrates. To monitor enzyme reaction by absorbance with a chromogenic substrate, the favorable wavelength is the longest difference absorbance peak of the chromogenic product versus the chromogenic substrate when the chromogenic product has the longest absorbance peak larger than that of the chromogenic substrate, or else, the favorable wavelength to monitor the reaction is the longest difference absorbance peak of the chromogenic substrate versus the chromogenic product. Of a pair of enzymes for SDESA, one enzyme is designated as Enzyme A if the favorable wavelength to monitor its reaction is larger than that to monitor the reaction of the other enzyme denoted as Enzyme B. Consequently, Enzyme A acts on Substrate A to give Product A while Enzyme B acts on Substrate B to yield Product B. The favorable wavelength to monitor the reaction of Enzyme A is λA and that of Enzyme B is λB (λA is longer than λB). The longest isoabsorbance peak of Substrate A is λ0 where Substrate A and Product A have the equal absorptivity. The following equations are derived on the basis of the assumption that the favorable wavelengths to monitor reactions of both enzymes are the longest difference absorbance peaks of the chromogenic products but still can be directly applied to the situation when the favorable wavelength(s) to monitor the reaction(s) of one or both enzymes is/are the longest difference absorbance peak(s) of the chromogenic substrate(s). To simplify resolving the overlapped absorbance of substances involved in SDESA, only the absorbance of two chromogenic substrates and two chromogenic products is considered. Assigning the two wavelengths under swift alternation to continuously monitor absorbance of reaction solution to λ1 and λ2 (λ1 is larger than λ2), the following symbols are defined for SDESA as follows. εA1: the difference in molar absorptivity of Product A versus Substrate A at λ1; εA2: the difference in molar absorptivity of Product A versus Substrate A at λ2; εB1: the difference in molar absorptivity of Product B versus Substrate B at λ1; εB2: the difference in molar absorptivity of Product B versus Substrate B at λ2; CPA: the instantaneous concentration of Product A; CPB: the instantaneous concentration of Product B; A1: the apparent instantaneous absorbance at λ1 of reaction solution; A2: the apparent instantaneous absorbance at λ2 of reaction solution; A10: the background absorbance at λ1 of the same reaction solution without any product; A20: the background absorbance at λ2 of the same reaction solution without any product; A1a: the interference-free difference in absorbance at λ1 of Product A versus Substrate A; A2b: the interference-free difference in absorbance at λ2 of Product B versus Substrate B. According to the linear additivity of absorbance and 1:1 stoichiometry for enzymatic conversion of a chromogenic substrate into chromogenic product, eqs 1 and 2 apply.

value on the actions of two enzymes and should satisfy the following two prerequisites: (a) the activity of either enzyme is free from any interference and (b) besides higher efficiency, another performance is comparable to or better than that by the separate assay under the same conditions. Hence, for SDESA, special methods are required to resolve the overlapped absorbance of substances, to estimate enzyme activities free from the inhibition/activation by any substance involved, and to have another performance comparable to that by a separate assay. For laboratory diagnosis, γ-glutamyltransferase (GGT) and lactate-dehydrogenase (LDH) in sera are usually measured so that SDESA of GGT and LDH has some significance.15−24 GGT action on γ-glutamyl-p-nitroaniline (GGPNA) produces p-nitroaniline (PNA) quantifiable by absorbance around 405 nm;25−28 reduced nicotinamide dinucleotide (NADH) as LDH substrate can be measured by absorbance around 340 nm. However, PNA competitively inhibits LDH; the linear range for measuring classical initial rates of GGT is unsatisfactory because GGT has a high Michaelis constant (Km) for GGPNA while GGPNA has limited solubility. SDESA of GGT and LDH is thus challenging but is successful using special methods to process absorbance of reaction solutions quantified via swift alternation of detection wavelengths. Moreover, alkaline phosphatase (ALP) and β-D-galactosidase (BGAL) are practical ELISA labels. ALP acts on 4-nitro-1-naphthylphosphate (4NNPP) to release 4-nitro-1-naphthol (4NNP) measurable by absorbance around 450 nm; BGAL acts on para-nitrophenyl-β-D-galacotoside (PNPG) to produce 4-nitrophenol (4NP) quantifiable by absorbance at 405 nm. SDESA of ALP on 4NNPP and BGAL on PNPG is simple because ALP has low Km for 4NNPP, and there is no interference from involved substrates/products with the action of either enzyme after the optimization of buffer composition. As a result, SDESA of ALP and BGAL is facile for concomitant ELISA of two components in one reaction well with ever-enhanced efficiency and another performance comparable to that via a separate assay. Herein, with GGT and LDH of sophisticated kinetics, we tested the proposed chemometrics and design issues of SDESA; with ALP and BGAL of simple kinetics, we demonstrated the facileness of the application of SDESA to concurrent ELISA of two components in one reaction well.

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EXPERIMENTAL SECTION Reagents and Animals. Noniondet P40 (NP-40), GGPNA, NADH, LDH from rabbit muscle (suspension in ammonium sulfate), calf intestinal alkaline phosphatase (ALP), and β-Dgalactosidase (BGAL) were from Sigma-Aldrich (St. Louis, MO, USA). para-Nitrophenyl-β-D-galactopyranoside (PNPG) was from BBI. 4-Nitro-1-naphthol (4NNP) was from Alfa Aesar (Tianjing, China). 4-Nitro-1-naphthylphospahate (4NNPP) was prepared by the reported procedure.29 Penicillin G and clenbuterol were from Dr. Ehrenstorfer GmbH. Mouse antipenicillin antibody (ab15070) and anticlenbuterol antibody (ab32005) were from Abcam. ReactiBind goat anti-Mouse IgG coated clear 96-well plates (No. 15134) were from Thermo Scientific. Other reagents were domestic products of analytical grade, unless otherwise stated. All reagents were used as received. Rabbits were from the Experimental Animal Center of Chongqing Medical University. Experiments with animals were approved by the Ethics Commissions of Chongqing Medical University, and efforts were made to alleviate animal suffering. Rabbit kidneys were homogenized with 100 mmol/L sodium phosphate buffer at pH 7.4 plus 0.1% NP-40 at 4 °C, and the supernatant after the removal of precipitates was kept at 4 °C until use in 8 h.28

A1 = A10 + εA1 × C PA + εB1 × C PB

(1)

A 2 = A 20 + εA2 × C PA + εB2 × C PB

(2)

When the differences in the absorptivity of each chromogenic product and its substrate is known at λ1 and λ2, the concentrations of Product A and B can be derived with eqs 1 and 2 in a group. However, it is laborious to determine so many spectral parameters required in eqs 1 and 2. A1a and A2b are proportional to product concentrations as described in eqs 3 and 4, respectively. A1a = εA1 × C PA

(3)

A 2b = εB2 × C PB

(4)

By defining R12 as the correction coefficient for the difference in absorptivity of Product A versus Substrate A at λ2 against that at λ1 2144

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20 s intervals until the change of A1 for Enzyme B reaction alone or A2 for Enzyme A reaction alone was over 0.005. Regression analysis of the changes of A2 on the changes of A1 during the reaction of Enzyme A alone gave the slope as the estimate of R12. Similarly, R21 was obtained via regression analysis of the changes of A1 on the changes of A2 during the reaction of Enzyme B alone. On the other hand, for the application of SDESA to ELISA, Biotek ELX 800 microplate reader was used with filters of 450 and 405 nm; the reader was controlled by Gene 5.0 software for simultaneously quantifying absorbance at 450 and 405 nm. Data were processed in the same way to estimate R12 and R21. Processing Reaction Data of GGT at a Low GGPNA Level. GGPNA has strong ultraviolet absorbance. For a reasonable range to quantify NADH absorbance at 344 nm, final GGPNA was set at 0.15 mmol/L but the linear range of classical initial rates of GGT was unsatisfactory. To avoid this disadvantage, an integration strategy is utilized to estimate initial rates by kinetic analysis of reaction process when enzyme activities are over the upper limit of linear response by the classical initial rate method.28,30−34 In brief, after the elimination of the interference of absorbance, A1a at 405 nm was processed with an executable software; when GGT activities exceeded the upper limit of linear response by the classical initial rate method, an improved integrated method was employed by kinetic analysis of reaction process to estimate Vm of GGT; this Vm was then converted into initial rate using a preset initial GGPNA concentration of 0.14 mmol/L.28 Km of rabbit kidney GGT for GGPNA was preset at 1.0 mmol/L while the inhibition constant of γ-glutamyl-glycinylglycine was preset at 185 μmol/L for the widest linear range by the integration strategy. Background absorbance (A10) was treated as a nonlinear parameter for estimation by kinetic analysis of reaction process. As an alternative to test the effect of the integration strategy on the quantification sensitivity of SDESA, the classical initial rates of GGT were converted into Vm using all the same kinetic parameters of GGT when they were below the upper limit of the classical initial rates; the response of GGT activities by Vm to GGT quantities was checked. Processing Competitive Inhibition of PNA on LDH. PNA exhibits competitive inhibition on LDH with an inhibition constant (Ki) of (24 ± 3) μmol/L (n = 3) at 3.0 mmol/L pyruvic acid (Figure S1, Supporting Information). Assuming the initial concentration of NADH is CB0, there is eq 9 with the same definitions of all symbols mentioned above. During the initial rate reaction, CB0 is a constant. After the integration of both sides in eq 9, there is eq 10 and thus eq 11.

by eq 5 and R21 as the correction coefficient for the difference in absorptivity of Product B versus Substrate B at λ1 against that at λ2 by eq 6, there are eqs 7 and 8, respectively. R12 = εA2 /εA1

(5)

R 21 = εB1/εB2

(6)

A1 = A10 + A1a + R 21 × A 2b

(7)

A 2 = A 20 + R12 × A1a + A 2b

(8)

By solving eqs 7 and 8 in a group, A1a and A2b are free of interference and serve as reaction curves of two enzymes in the same reaction solution. With simultaneous assays of A1 and A2 of one reaction solution during the action of Enzyme A from a sample on Substrate A alone so that there is no absorbance from Product B and Substrate B, regression analysis of the changes of A2 on the changes of A1 gives the slope as an estimate of R12. Similarly, regression analysis of the changes of A1 on the changes of A2 of a reaction solution under the action of a sample of Enzyme B on Substrate B alone gives the slope as an estimate of R21. To treat the interference from the inhibition by any product/substrate involved in SDESA, A10 and/or A20 are required to calculate the levels of substrates/products. However, A10 and/or A20 can be treated as parameter(s) for nonlinear fitting instead of being determined by experimentation. Estimation of the Longest Isoabsorbance Wavelength λ0. For SDESA of GGT plus LDH, GGT serves as Enzyme A while LDH is Enzyme B. Reactions of GGT and LDH were carried out in 1.00 mL of 100 mmol/L PBS at pH 7.4. GGT reaction solution contained a final concentration of 0.15 mmol/L GGPNA and 75 mmol/L glycinylglycine; LDH reaction solution contained a final concentration of 0.15 mmol/L NADH and 3.0 mmol/L pyruvic acid, and reaction solutions of the mixture of GGT and LDH contained the final levels of all the substrates used for assays of GGT and LDH. These substrate levels were used throughout this study, unless otherwise stated. Absorption spectra of reaction solutions of GGT alone, LDH alone, and mixed GGT and LDH were recorded separately with a Shimadzu UV 2550 at 0.5 nm step. From those absorbance spectra, the longest isoabsorbance wavelength (λ0) where GGPNA and PNA have the equal absorptivity was estimated for SDESA of GGT and LDH. For SDESA of ALP and BAGL, ALP serves as Enzyme A while BGAL is Enzyme B. For SDESA, 50 mmol/L Tris−HCl at pH 7.5 plus 50 μmol/L citrate was used at 25 °C. Similarly, the absorbance spectra of reaction solutions of ALP on 0.15 mmol/L 4NNPP alone, BGAL on 6.0 mmol/L PNPG alone, and the mixture of ALP and BGAL on the mixture of the two chromogenic substrates at the same concentrations in the same buffer were recorded separately at 0.5 nm step. λ0 was estimated from those absorbance spectra for SDESA of ALP and BGAL. Estimation of the Correction Coefficients R12 and R21. Mapada UV 1600 PC spectrophotometer was linked to a computer running M.Wave Professional 2.0 (http://www.mapada.com.cn/ showcp.php?id=27) for measuring absorbance at one or two wavelengths, unless otherwise stated. To estimate correction coefficients R12 and R21, required substrate(s) of any enzyme among GGT, LDH, ALP, and BGAL was/were mixed and incubated at (25.0 ± 0.5) °C for 10 min before the initiation of reaction by the addition of an enzyme sample. Thirty seconds later, the absorbance at 405 nm (A1) and that at 344 nm (A2) for GGT or LDH and the absorbance at 450 nm (A1) and that at 405 nm (A2) for ALP or BGAL were recorded concomitantly at

dA 2b = εB2 × C B0 × Vm ×

dt K m × (1 + A1a /K i /εA1) + C Bo (9)

∫0

t

dA 2b = εB2 × C B0 × Vm ×

∫0

t

1 × dt K m × (1 + A1a /K i /εA1) + C B0 (10)

X=

∫0

Vm =

t

∫0

1 × dt K m × (1 + A1a /K i /εA1) + C B0

(11)

dA 2b /(εB2 × C B0 × X )

(12)

t

V2 = (0.93 × C B0 × Vm)/(K m + 0.93 × C B0) 2145

(13)

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mg on 6.0 mmol/L PNPG in the Tris−HCl buffer at 25 °C. Finally, the tracer was diluted with the Tris−HCl buffer to 6.0 U per mL containing 3% glycerol and 0.02% NaN3 for storage at 4 °C prior to use. Preparation of Mimicked Milk Samples. A pool of milk drinks from a nearby supermarket after centrifugation at 12 000 rpm for 20 min to remove most of the insoluble components was used as the blank of milk sample. For constructing response curves of binding ratios to logarithmic hapten quantities by ELISA, clenbuterol and penicillin G with known quantities varying over three magnitudes but possessing a fixed quantity ratio were added to aliquots of the blank. To determine the recovery of either hapten by ELISA, clenbuterol and penicillin G with known quantities varying within their quantifiable ranges but bearing different quantity ratios were added to aliquots of the blank. To test the reliability of the application of SDESA to ELISA, clenbuterol and penicillin G with unknown quantities exceeding their quantifiable ranges and possessing random quantity ratios were added to aliquots of the blank. Competitive ELISA of Clenbuterol and Penicillin G via SDESA and Separate Assay. Biotek ELX 800 microplate reader was employed to measure absorbance concurrently at 450 and 405 nm via swift alternation of two filters in an isolated small room air-conditioned at 25 °C. The Tris−HCl buffer was kept in a water bath at 25 °C. The complexes of each tracer and mouse antihapten antibody were captured by immobilized goat antimouse IgG polyclonal antibodies (Scheme 1). Anticlenbuterol

Presetting A1a as a constant within a short sampling interval, numerical integration of eqs 10 and 11 gives eq 12. Presetting NADH at 0.14 mmol/L, the intrinsic initial rate of LDH (V2) is derived from Vm.28,30−34 Clearly, A20 is not required for the integration of the left side in eq 10, but A10 is required for the integration of the right side in eqs 10 or 11. This approach to LDH activity applies to data with NADH consumption below 20%. Comparison of SDESA against Separate Assay. One unit of any enzyme is the quantity of the enzyme to consume one micromole of substrate or produce one micromole of product per min under the stated conditions. SDESA and a separate assay were carried out with the same final substrate levels in the same buffers as described above. λ1 and λ2 were 405 and 344 or 340 nm for SDESA of GGT and LDH and 450 or 460 nm and 405 or 404 nm for SDESA of ALP and BGAL, respectively. The total time to record absorbance at two wavelengths was within 10.0 min. The reaction mixture for SDESA of GGT and LDH contained four substrates including GGPNA plus glycinylglycine for GGT and NADH plus pyruvic acid for LDH. The reaction mixture for SDESA of ALP and BGAL contained 4NNPP and PNPG. All independent data were determined in triplicate at least. Response curves of activities (initial rates) to enzyme quantities were examined to estimate the quantification limits of either enzyme by SDESA at different activities of the other enzyme; the samples with GGT and LDH mixed at 1:1, 1:5 or 5:1, and 1:25 or 25:1, or with ALP and BGAL mixed at comparable ratios, were employed. For a separate assay of either enzyme, just its own substrate(s) was/were employed at the same final concentration(s) while other assay conditions and the samples were completely the same. Application of SDESA to ELISA of Two Components in One Reaction Well. Conjugation of Clenbuterol to ALP. Clenbuterol (0.14 g) in 160 μL of 0.10 mol/L aqueous HCl was mixed with a 25 μL solution of 0.17 mol/L sodium nitrite for reaction of 30 min at 25 °C to yield diazotized clenbuterol. Then, a 100 μL solution of ALP at 4.0 mg per mL in 0.20 mol/L sodium borate buffer at pH 9.2 was added for reaction of 120 min at 4 °C.35 The modified ALP was passed through a Sephadex G25 column (GE healthcare, 10 mm × 150 mm) equilibrated and eluted with 50 mmol/L Tris−HCl buffer plus 50 μmol/L citrate at pH 7.5 (the Tris−HCl buffer was used throughout the experiments of ELISA, unless otherwise stated); the first peak of absorbance at 280 nm in a total of 2.0 mL was collected. Clenbuterol-modified ALP as the tracer had the specific activity of 60 U per mg on 4NNPP at 0.15 mmol/L in the Tris−HCl buffer at 25 °C and was diluted with the Tris−HCl buffer to a final 3.0 U per mL solution containing final 3% glycerol and 0.02% NaN3. The diluted solution of clenbuterol-modified ALP was stored at 4 °C prior to use in 1 week. Conjugation of Penicillin G to BGAL. Penicillin G (0.73 g) was activated with 0.40 g of 1-ethyl-(3-dimethylaminopropanyl)-carbadiimide and 0.30 g of 1-hydroxyl-benzotriazole for 30 min at 25 °C in 2.0 mL N,N′-dimethylformamide; the resulting yellow solution was diluted by 1:14 with 50 mmol/L sodium phosphate buffer at pH 7.5. The diluted solution of 50 μL of the activated penicillin G was mixed with 0.44 mg of BGAL in 250 μL of phosphate buffer (50 mmol/L at pH 7.5).36 After reaction for 120 min at 4 °C, modified BGAL was purified through a Sephadex G25 column (10 mm × 150 mm), equilibrated, and eluted with the Tris−HCl buffer; the first peak in a total of 2.0 mL by absorbance at 280 nm was collected. Penicillin G-modified BGAL as the tracer had a specific activity of 30 U per

Scheme 1. Procedure to Apply SDESA to ELISA of Two Haptens in One Reaction Well

antibody and antipenicillin antibody were diluted by 1:5000 and 1:500, respectively, and 50 μL of each antibody was used in every well. After capturing reaction for 60 min under mild continuous shaking, the wells were washed for three times, each with 200 μL of 50 mmol/L Tris−HCl at pH 7.5 plus 0.05% Tween 20. Two tracer solutions and each sample after dilution with the Tris− HCl buffer were mixed at 1:1:3, and 100 μL of the mixture was transferred into a well for competitive binding. After reaction for 60 min under mild continuous shaking, the wells were washed for three times, each with 200 μL of 50 mmol/L Tris−HCl at pH 7.5 plus 0.05% Tween 20. For ELISA via SDESA, two bound tracers were quantified concomitantly with a 200 μL solution of 0.15 mmol/L 4NNPP and 6.0 mmol/L PNPG in the Tris−HCl buffer. For ELISA via a separate assay, a 200 μL substrate solution of either 0.15 mmol/L 4NNPP alone or 6.0 mmol/L PNPG alone in the Tris−HCl buffer was used. Absorbance at 450 and 405 nm was concurrently recorded in 40 min at 5.0 min intervals 2146

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The solution to the first problem needs a suitable pair of chromogenic substrates, a proper pair of λ1 and λ2 for quantifying absorbance, and a special chemometrics method to mine the interference-free absorbance of chromogenic products/substrates. The method for mining the interference-free absorbance of Product A and B from the apparent absorbance at λ1 and λ2 is described in eqs 7 and 8 based on the linear additivity of absorbance; it is universally applicable to any pair of λ1 and λ2. To enhance the performance for quantifying two chromogenic products by absorbance at two wavelengths under swift alternation, R21 in eq 7 and R12 in eq 8 should be as negligible as possible, and thus, a suitable pair of chromogenic substrates together with a proper pair of λ1 and λ2 is defined as follows. Of a reaction solution, the absorbance at λ0, where Product A and Substrate A have the same absorptivity, will not be altered by the action of Enzyme A alone. In this case, Product B can be quantified by the absorbance at λ0 with a limit comparable to that by a separate assay at λ0, and even with the comparable sensitivity if λB is equal or close to λ0. Inevitably, the range to quantify Product B absorbance is narrower if Substrate A has strong absorbance at λ0. Meanwhile, if λA is much larger than λB, the absorbance of Product B at λA will not ruin the sensitivity, limit, and linear range for quantifying Product A by the absorbance at λA or any suitable wavelength nearby λA. Clearly, the smaller distance from λ0 to λB yields a tinier R12 to improve the sensitivity and limit for quantifying Product B; the larger distance from λA to λB gives a smaller R21 to mitigate the impacts of Enzyme B action on the limit to quantify Product A. Therefore, a suitable pair of chromogenic substrates should produce λB close to λ0 but much shorter than λA; as for a proper pair of wavelengths, λ1 can be close to λA but λ2 is preferable to be equal to λ0. The second problem is solved by the estimation of Vm of either enzyme as the intrinsic activity free from the inhibition/ activation by any substance involved in SDESA. It is ideal to avoid the interference with the action of either enzyme during SDESA by any suitable means like the use of coupled reactions, optimization of reaction conditions, or buffer compositions;37 when such interference with either enzyme is inevitable, the interference can be eliminated by estimating Vm of the enzyme via kinetic analysis of reaction curve. A1a and A2b free of interference are reaction curves of Enzymes A and B, respectively. For a single-enzyme reaction, kinetic analysis of reaction curve estimates Vm that is free from the interference by its substrates/ products.3−6,28,30−34,37−40 In detail, Vm for a single-enzyme reaction is estimated by fitting an explicit integrated rate equation with the predictor variable of reaction time to reaction curve of interest after data transformation or numerical integration of differential rate equation(s) to yield a series of calculated reaction curves for fitting to reaction curve of interest.28,30−34,37−40 Clearly, reaction kinetics of either enzyme under the interference by the substrates/products of the other enzyme during SDESA is too complex to have an explicit integrated rate equation with reaction time as the predictor variable. In this case, the estimation of Vm of either enzyme by kinetic analysis of reaction curve requires numerical integration of the differential rate equation to yield calculated reaction curves for fitting to reaction curve of interest and is applicable to reaction curves with different substrate consumption percentages.31 When the consumption percentages of substrate are restricted for initial rate reaction, a simplified fitting as eqs 11 and 12 can estimate Vm. To index enzyme activity, Vm can be directly employed or be converted into initial rate at a preset substrate level as described below. Hence, the estimation of Vm via

after 5.0 min quick shaking of ELISA plates on QilinBeier QB9001 shaker. Other Methods for Data Processing. Mean and standard deviation (SD) of initial rates (absorbance change rates) of the reagent blank were determined from more than 11 independent assays. Coefficient of variation (CV) was the percentage of SD to the mean. Limit of detection (LOD) of each enzyme was the lowest quantity of the enzyme (initial rate) giving rise to instrumental signal change higher than that of the reagent blank plus three-times SD of the reagent blank. Limit of quantification (LOQ) of each enzyme was the lowest enzyme quantity producing instrumental signal change no less than the sum of LOD and five times of the standard error of estimate for the response of activities (initial rates) to enzyme quantities. The quantification sensitivity of an enzyme is the response slope of activities to quantities of the enzyme. The upper limit was the highest activity showing deviation from the previous linear response plot no more than twice the standard error of estimate. For processing A1a under the complicated inhibition by GGT products at a limited GGPNA level, the same software for the integration strategy was used with parameters stated above.28,30−34 To process the inhibition of PNA on LDH, numerical integration and calculation were preformed with functions incorporated in MS Excel 03. For competitive ELISA (Scheme 1), the binding ratio of either tracer was the activity (net change of A1a or A2b over the reaction period of 40 min) of the tracer bound in the presence of a hapten as a competitor divided by the activity of the tracer bound in the absence of the competitor (the net change of A1a or A2b over 40 min in the absence of any competitor was higher than 0.280 but smaller than 0.310 throughout the study). The response of binding ratios to logarithmic quantities of either hapten was examined. LOD of a hapten was its lowest quantity to cause a reduction in the binding ratio of the tracer no less than three times of SD of the binding ratios in the absence of any competitor. LOQ of a hapten was its lowest quantity to produce a binding ratio of the tracer within the range for linear response of binding ratios to logarithmic hapten quantities. The upper limit of quantification of a hapten was its largest quantity to produce a binding ratio of the tracer within the range for linear response of binding ratios to logarithmic quantities of the hapten. Recovery of ELISA via SDESA or a separate assay was the percentage of the quantity of a hapten estimated to that added to the milk sample. The percentage of quantity of each hapten in a milk sample determined by ELISA via SDESA to that via a separate assay was calculated to examine consistency between two methods.

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RESULTS AND DISCUSSION Theoretical Considerations. For the concomitant satisfaction to those two prerequisites of SDESA, the following technical problems specific for SDESA should be solved sequentially: (a) the quantification of Product A and B by the absorbance at λ1 and λ2, respectively, is free from the interference of overlapped absorbance of any substance and has the sensitivity, limits, and linear ranges comparable to those by a separate assay, correspondingly, (b) the estimated activity of either enzyme is free from the inhibition/activation by any substance involved in the reactions of two enzymes of interest during SDESA, and (c) the assay of the activity of either enzyme by SDESA has the sensitivity, limit, and linear range comparable to those by a separate assay via any practical method under the same conditions, respectively. The solutions to the three problems are the design issue of SDESA and are described below. 2147

dx.doi.org/10.1021/ac302786p | Anal. Chem. 2013, 85, 2143−2154

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GGPNA at 340 nm was about 1.13-fold of that at 344 nm; for the same maximum absorbance, the maximum level of NADH measurable at 340 nm was reduced by about 5% in comparison to that at 344 nm. Hence, GGPNA and NADH is a suitable pair for SEDSA of GGT and LDH; λ1 can be preset at 405 nm while λ2 is preferable to be 344 nm.

kinetic analysis of reaction curve provides enzyme activity free of the interference of substances involved in SDESA. The third problem is solved using an integration strategy to estimate the activity of Enzyme A if the levels of Substrate A are lower than Km. When a suitable pair of Substrates A and B is used, the quantification limits of both enzymes by SDESA can be comparable to those by separate assay under the same conditions. However, for a reasonable range to measure the absorbance of Product B at λ0, the level of Substrate A is limited; this limitation negatively impacts the linear range and sensitivity to quantify classical initial rates of Enzyme A of high Km. Alternatively, the quantification sensitivity of either enzyme by SDESA can be the same as that by a separate assay if Vm of either enzyme is estimated via an improved integrated method, but the efficiency at lower enzyme activities is horrible because of the requirement of long reaction time to record reaction data with sufficient percentages of substrate consumption for analysis.5,6,37−40 Fortunately, we developed an integration strategy to measure enzyme activities with a wide linear range and reasonable efficiency at substrate levels from 0.1-fold Km to 10-fold Km.28,30−34 In the integration strategy, (a) classical initial rate is estimated when enzyme activity is not higher than the upper limit of linear response by the classical initial rate method; (b) Vm is estimated via kinetic analysis of reaction curve when enzyme activity is higher than the upper limit of linear response by the classical initial rate method; (c) Vm is converted into initial rate, or vice versa, according to known kinetics at substrate levels of 93% of the initial values.28,30−34 The conversion of initial rates by SDESA into Vm can give the same sensitivity as by a separate assay. The integration strategy prefers the final level of the limiting substrate comparable to Km; it is the sole alternative for measuring enzyme activities with a wide linear range at substrate levels comparable to Km. For SDESA, Substrate B can be preset at levels for reasonable linear ranges of classical initial rates. Hence, the use of the integration strategy provides a wide linear range of activities of Enzyme A by SDESA at levels of Substrate A lower than Km; the classical initial rate method alone is effective when levels of Substrate A are higher than Km and SDESA prefers Enzyme A of low Km for Substrate A. Taken together, with a suitable pair of chromogenic substrates and proper methods to process data, SDESA is promising. Complicated substrate/product inhibition kinetics of GGT, high Km of GGT for GGPNA but limited final levels of GGPNA due to low solubility, and the inhibition of LDH by PNA as GGT product greatly challenge SDESA of GGT and LDH.28 However, low Km of ALP for 4NNPP and the elimination of the activation of BGAL by ALP product via the optimization of buffer compositions simplify SDESA of ALP and BGAL as ELISA labels. Hence, SDESA is tested with those two models separately. Selection of λ1 and λ2 and Estimation of R12 and R21. The action of GGT alone on GGPNA produced λ0 close to 344 nm (Figure 1). Molar absorptivity of NADH at 344 nm is over 95% of that at 340 nm. By presetting λ2 at λ0 while λ1 at λA to simultaneously quantify A1 and A2 of reaction solution containing only GGPNA and glycinylglycine, regression analysis of the changes of A2 on that of A1 gave R12 of (0.012 ± 0.001) (n = 4). Similarly, regression analysis of the changes of A1 on the changes of A2 of reaction solution containing only NADH and pyruvic acid gave R21 of (0.0082 ± 0.0011) (n = 4). When λ2 was preset at 340 nm while λ1 was preset at 405 nm, however, R12 was (−0.198 ± 0.002) (n = 4), nearly 16-fold higher than that at 344 nm. Meanwhile, R21 was (0.0078 ± 0.0010) (n = 4) (Figure S2, Supporting Information). When GGPNA was fixed at 0.15 mmol/L, the absorbance of

Figure 1. Change of absorbance spectra of enzyme reaction solutions of GGT and LDH. Lines from 1 to 4 indicated the reaction duration of 0, 2, 4, 6 min, respectively. (a) GGT alone; (b) LDH alone; (c) GGT and LDH.

The action of ALP on 4NNPP produces 4NNP with λ0 of about 405 nm and λA close to 458 nm; the absorptivity of 4NNP at 450 nm was over 92% of that at 460 nm. BGAL action on PNPG produces 4NP with λB close to 405 nm (Figure 2). When λ2 was preset at 405 nm while λ1 was set at 450 nm on the spectrophotometer, R12 estimated with 4NNPP alone was (−0.0060 ± 0.0003) (n = 5), but R21 estimated with PNPG alone was (0.1813 ± 0.0013) (n = 5); when λ2 was 405 nm while λ1 was 460 nm, R12 with 4NNPP alone was (−0.0056 ± 0.0003) (n = 5), but R21 with PNPG alone became (0.0799 ± 0.0020) (n = 5). The results support that λ1 distant from λ0 is preferable. The setting of λ2 at 404 nm led to R12 as large as (−0.0219 ± 0.0015) (n = 5) while R21 of (0.1799 ± 0.0014) (n = 5) for λ1 at 450 nm and R12 of (−0.0205 ± 0.0014) (n = 5) while R21 of (0.0792 ± 0.0024) (n = 5) for λ1 at 460 nm (Figure S3, Supporting Information). The results support that λ2 is preferable to be 405 nm. 2148

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on GGT, and the inhibition of LDH by PNA as GGT product, all make SDESA of GGT and LDH a challenging model to test the proposed chemometrics for data processing and the design issue of SDESA. LDH displays Km of (38 ± 2) μmol/L (n = 3) for NADH (Figure S1, Supporting Information). Initial NADH concentration was about four-times Km of LDH, and the linear decrease in absorbance before 20% consumption of NADH was expected. During the concomitant actions of GGT and LDH, the change of the absorbance at 344 nm of NADH displayed no significant deviation from linear decrease. However, reaction rates indexed by changes of A2b always showed small negative deviation from that by separate assay (Figure 3a). By SDESA, the linear range

Figure 2. Change of absorbance spectra of enzyme reaction solutions of ALP and BGAL at 0.10 mmol/L 4NNPP and 0.4 mmol/L PNPG. Lines from bottom to top indicated reaction time of 0, 1, 3, 5, 7 min, respectively. (a) ALP alone; (b) BGAL alone; (c) ALP and BGAL.

Using Biotek ELX 800 microplate reader with two filters at 405 and 450 nm to measure the absorbance, R12 was (−0.0064 ± 0.0004) (n = 5) and R21 was (0.1796 ± 0.0025) (n = 5) (Figure S4, Supporting Information). Those correction coefficients clearly support the criteria to select the two wavelengths for enhancing SDESA and the feasibility to test the application of SDESA to ELISA with conventional microplate readers. Additionally, those correction coefficients for ALP and BGAL with two wavelengths within the visible region display higher precision; the lower precision of correction coefficients with GGT and LDH can be attributed to the alternation of light sources from ultraviolet to visible region. Thus, 4NNPP and PNPG is a favorable pair for SDESA of ALP and BGAL; λ1 can be preset at 450 nm while λ2 is preferable to be 405 nm for SDESA. SDESA of GGT and LDH: A Challenging Model. Clearly, it was easy to record the changes of absorbance at 344 nm and that at 405 nm under the concomitant actions of LDH and GGT with their activities at dynamic ratios (Figure S5, Supporting Information). As expected from tiny R12 and R21, the correction of the overlapped absorbance among chromogenic substances caused small changes in A1a and A2b. However, the limitation on final GGPNA levels, complicated substrate/product inhibition

Figure 3. Correction of absorbance (a) and response of initial rates to LDH quantities (b). (a) SDESA, absorbance not corrected, A344 = −0.0617 × t + 1.4874, R2 = 0.9954 SDESA, absorbance corrected, A344 = −0.0624 × t + 1.4845, R2 = 0.9965. Separate assay, A344 = −0.0711 × t +1.4581, R2 = 0.9989. (b) SDESA, absorbance and PNA inhibition corrected, ΔA/min = 1.1155 × χ + 0.0026, R2 = 0.9935. Separate assay, ΔA/min =1.1715 × χ + 0.0016, R2 = 0.9943.

for the classical initial rates of LDH was narrower than that by a separate assay at the same levels of substrates, and the slope of linear response was slightly smaller (Figure 3b). The upper limit of linear response of the classical initial rates of LDH was even smaller when GGT activities were higher; the correction of the overlapped absorbance only slightly improved the upper limit of linear response. These results indicated that LDH may be inhibited by substrates/products of GGT. Indeed, PNA inhibited LDH competitively against NADH with an inhibition constant of (24 ± 3) μmol/L (n = 3), but all the substrates of GGT showed negligible actions on LDH. After the correction of the inhibition 2149

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of PNA on LDH by estimating Vm via kinetic analysis of reaction curve, the linear range for quantifying initial rates of LDH by SDESA was expanded by nearly three-times (Figure 3b). Under these conditions, the linear range for quantifying LDH was comparable to that by a separate assay, but the sensitivity was about 96% of that by a separate assay due to effects of unknown factors. On the basis of the slope of linear response, the quantification sensitivity of LDH by absorbance at 340 and 344 nm had a tiny difference. Hence, the estimation of Vm is effective to eliminate PNA inhibition on LDH. At the limited level of GGPNA, the linear range to quantify the classical initial rates of GGT was quite narrow (Figure 4a). By

assay at the same level of GGPNA, supporting no interference with GGT action from substrates/products of LDH. Certainly, GGPNA levels for separate assay of GGT can be 2.0 mmol/L, but the linear range by the integration strategy at 0.15 mmol/L GGPNA was wider than that by the classical initial rate method at 2.0 mmol/L GGPNA.28 When classical initial rates were converted into Vm in the integration strategy, the linear range was the same (Figure 4b). Hence, the use of the integration strategy for SDESA of GGT at a GGPNA level much lower than Km provides a linear range and quantification sensitivity of GGT activities comparable to those via a separate assay, respectively. When activities of GGT and LDH were very low, the LOQ of either enzyme was comparable to that by the separate assay. Without the correction of absorbance from NADH at 405 nm, the LOQ of GGT was still comparable to that by the separate assay when LDH activities were varied from 1:1 to 25:1 of GGT (Table 1). On the other hand, GGT action on GGPNA caused small changes of the absorbance at 344 nm, but the LOQ of LDH after only correction of such absorbance was higher than that by the separate assay at the same NADH level when GGT activities were 25-fold higher. The elimination of the competitive inhibition of LDH by PNA provided the LOQ of LDH comparable to that by a separate assay at the same NADH level when GGT activities were varied from 1:1 to 25:1 of LDH. When λ2 was preset at 340 nm, LOQ of LDH was about 1.4-fold higher than that with λ2 preset at 344 nm; this difference should be due to much larger R12 and supports the preference of the setting of λ2 at λ0 for SDESA. Therefore, the chemometrics, the criteria to select a suitable pair of chromogenic substrates and a proper pair of wavelengths for quantifying the absorbance, are effective for SDESA. These results suggest the practice of SDESA of GGT and LDH in sera for their diagnostic roles. However, SDESA of GGT and LDH requires a final GGPNA level much lower than Km and, thus, a long time to record the reaction curve but reduce the overall efficiency. The application of SDESA to ELISA is more absorbing and SDESA of ALP and BGAL under optimized conditions is facile. Therefore, the application of SDESA to ELISA is further tested. SDESA of ALP and BGAL: A Simple Model Applicable to ELISA. For applying SDESA to ELISA, the primary prerequisite is the spectral properties of a pair of chromogenic substrates for two enzymes as candidate labels because these spectral properties principally determine the detection and quantification limits by applying SDESA to ELISA. Absorbance at wavelengths over 400 nm is measurable with filters on conventional microplate readers; the action of Enzyme A on Substrate A should thus produce λ0 no less than 400 nm. ALP and BGAL have good stability; spectral properties of 4NNPP and PNPG as their chromogenic substrates are satisfactory for SDESA with conventional microplate readers. Hence, the applicability of SDESA to ELISA is tested with ALP and BGAL as labels. An optimized buffer system is selected for SDESA of ALP and BGAL at first. After intensive screening, 50 mmol/L Tris−HCl buffer at pH 7.5 was tested for SDESA of ALP and BGAL. In this buffer, BGAL had Km of 1.7 mmol/L for PNPG, but ALP had Km of (7.9 ± 2.0) μM for 4NNPP (n = 3), supporting the classical initial rate method alone should be effective to SDESA of ALP and BGAL. No products/substrates involved in SDESA inhibit ALP or BGAL. However, BGAL is activated by phosphate as ALP product (Figure S6, Supporting Information); a complicated method to process data limits the routine application

Figure 4. Response by SEDSA of (a) initial rates and (b) Vm to GGT quantities. (a) SDESA of GGT via the integration strategy, ΔA/min = 2.6565 × χ − 0.0061, R2 = 0.9965. Separate assay via the integration strategy, ΔA/min = 2.6971 × χ − 0.0059, R2 = 0.9965. (b) SDEDSA of GGT via the integration strategy, Vm = 340.92 × χ −0.779, R2 = 0.996. SDEDSA via the integration strategy, Vi = 269.15 × χ −0.615, R2 = 0.996.

an integration strategy employing kinetic analysis of reaction curve to estimate Vm and convert such Vm into initial rates at high enzyme activities, the linear range of GGT initial rates by SDESA was expanded by nearly four times. Due to different sources of GGT, the optimal kinetic parameters for the integration strategy to estimate Vm of rabbit kidney GGT displayed small differences from those of rat kidney GGT.28 On the basis of the slope of linear response, the sensitivity to quantify GGT initial rates by SDESA showed negligible difference from that by a separate 2150

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Table 1. Comparison of Quantification Limits by SDESA of GGT and LDH and a Separate Assaya conditions (n = 7)

a b

ΔA in 2 min

samples

ΔA in 2 min

GGT:LDH (1:1)

ΔA in 2 min

GGT:LDH (5:1)

ΔA in 2 min

GGT:LDH (25:1)

ΔA in 2 min

GGT:LDH (1:5)

ΔA in 2 min

GGT:LDH (1:25)

340 nm

344 nm

405 nm

wavelength methods

SDESA

SAb

SDESA

SAb

SDESA

SAb

mean SD LOD STEYX LOQ STEYX LOQ STEYX LOQ STEYX LOQ STEYX LOQ

−0.0003 0.0017 −0.0053 0.0043 −0.0269 0.0046 −0.0284 0.0031 −0.0206

−0.0003 0.0017 −0.0053 0.0023 −0.0170 0.0027 −0.0186 0.0021 −0.0161

−0.0006 0.0011 −0.0040 0.0022 −0.0149 0.0032 −0.0201 0.0024 −0.0161

−0.0006 0.0011 −0.0037 0.0020 −0.0136 0.0025 −0.0162 0.0019 −0.0133

−0.0004 0.0005 0.0012 0.0022 0.0120

−0.0004 0.0005 0.0013 0.0015 0.0088

0.0003 0.0026 0.0003 0.0027

0.0005 0.0039 0.0004 0.0032

The definitions of LOD and LOQ are described in the text. STEYX was the standard error of estimate for regression analysis of the linear response. SA: separate assay.

of SDESA to ELISA. The absorptivity of 4NNP at 450 nm is over 25 (mmol/L)−1·cm−1 at pH 7.5.41 For the increase in absorbance at 450 nm below 1.300, the maximal concentrations of 4NNP and phosphate released from 4NNPP in reaction solutions should be smaller than 60 μmol/L. In 50 mmol/L Tris−HCl buffer at pH 7.5, citrate is an activator of BGAL but a weaker inhibitor of ALP. The addition of final 50 μmol/L citrate caused negligible inhibition on ALP but increased BGAL activity by about 10% and eliminated the activation of BGAL by phosphate below 60 μmol/L. In 50 mmol/L Tris−HCl at 7.5 plus final 50 μmol/L citrate, ALP was about 90 U per mg on 0.15 mmol/L 4NNPP while BGAL was about 36 U per mg on 6.0 mmol/L PNPG. Hence, 50 mmol/L Tris−HCl at 7.5 plus 50 μmol/L citrate was used for SDESA of ALP and BGAL. After the correction of overlapped absorbance, SDESA of ALP and BGAL with spectrophotometer was robust (Figure 5a,b). LOD, LOQ, and linear range of initial rates of either ALP or BGAL were comparable to those by a separate assay, respectively; when activities of the counterpart enzyme were varied from 1:1 to 27:1; the setting of λ2 at 404 nm gave LOQ usually more than 3-fold of that by setting λ2 at 405 nm, supporting the preference of the setting of λ2 at λ0 for SDESA (Table S1, Supporting Information). On Biotek ELX 800 microplate reader, SDESA of ALP and BGAL produced LOD and LOQ of either enzyme about 1.4-fold of those by a separate assay, correspondingly, when activity ratios of two enzymes were varied from 1:1 to 1:27 (Table 2). Hence, SDESA of ALP on 4NNPP and BGAL on PNPG may be applicable to ELISA of two components in one reaction well. By concurrent ELISA of clenbuterol and penicillin G in one reaction well via SDESA, the response of binding ratios to logarithmic quantities of either hapten was similar to that via a separate assay under the same conditions (Figure 6). CVs for binding ratios by ELISA via SDESA or a separate assay were within 6% (Table 3). LOD, LOQ, and quantifiable range of either hapten by ELISA via SDESA under the same conditions were comparable to those via a separate assay, correspondingly (Table S2, Supporting Information). The average recoveries of clenbuterol and penicillin G in milk samples at tested levels by ELISA via SDESA were (97% ± 8%) and (103% ± 9%), respectively; average recoveries of clenbuterol and penicillin G in milk by

Figure 5. Comparison of responses of classical initial rates to quantities of ALP and BGAL by SDESA and a separate assay. (a) Comparison of response of classical initial rates to quantities of ALP. (b) Comparison of response of classical initial rates to quantities of BGAL. 2151

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Table 2. Comparison of Quantification Limits of ALP and BGAL by SDESA and a Separate Assay on Biotek ELX 800 Microplate Readera conditions (n = 7)

a b

ΔA in 40 min

system fluctuation

ΔA in 40 min

ALP:BGAL (1:1)

ΔA in 40 min

ALP:BGAL (1:3)

ΔA in 40 min

ALP:BGAL (1:9)

ΔA in 40 min

ALP:BGAL (1:27)

ΔA in 40 min

ALP:BGAL (3:1)

ΔA in 40 min

ALP:BGAL (9:1)

ΔA in 40 min

ALP:BGAL (27:1)

450 nm

405 nm

wavelength methods

SDESA

SAb

SDESA

SAb

mean SD LOD STEYX LOQ STEYX LOQ STEYX LOQ STEYX LOQ STEYX LOQ STEYX LOQ STEYX LOQ

0.023 0.001 0.025 0.012 0.083 0.004 0.040 0.006 0.043 0.007 0.051

0.021 0.001 0.023 0.014 0.091 0.001 0.028 0.002 0.033 0.004 0.041

−0.003 0.002 0.003 0.043 0.217

−0.002 0.002 0.002 0.019 0.095

0.011 0.045 0.014 0.048 0.010 0.051

0.008 0.042 0.009 0.040 0.007 0.042

The definitions of LOD and LOQ are described in the text. STEYX was the standard error of estimate for regression analysis of the linear response. SA: separate assay.

ELISA via a separate assay were (97% ± 4%) and (101% ± 5%), correspondingly (Table 3). The percentages of hapten quantities in milk samples determined by ELISA via SDESA to those determined by ELISA via a separate assay were (100 ± 6%) and (99 ± 5%) for clenbuterol and penicillin G, respectively (Table S3, Supporting Information). LOD and LOQ of clenbuterol by SDESA or a separate assay under the same conditions were lower than those of penicillin G, respectively. These differences in performance of ELISA of clenbuterol and penicillin G should be attributed to the use of larger quantities of both the tracer and the monoclonal antibody against penicillin G. Therefore, SDESA is applicable to ELISA. Notably, CVs and LOQ of quantities of clenbuterol or penicillin G by ELISA via SDESA or a separate assay under the same conditions were larger than those reported by ELISA via a separate assay under the optimum conditions for measuring either tracer alone.35,36,41 The range from the upper limit to LOQ of clenbuterol by ELISA via SDESA or a separate assay under the same conditions was consistently about 500-fold, nearly 30-fold wider than that by ELISA via a separate assay under the optimum conditions for ALP.35 The quantifiable range for penicillin G is about two magnitudes, still wider than that by ELISA via a separate assay under optimum conditions for BGAL.36,41 Under the assay conditions suitable for SDESA of ALP and BGAL, the maximum catalytic capacity of ALP is less than 2% of that under optimum conditions for measuring ALP alone and that of BGAL is below 10% of that under optimum conditions for measuring BGAL alone (http://www. sigmaaldrich.com, catalogue no. P0114 and G4155). The low activity of a tracer results in the needs of much larger quantities of both its antibody and the tracer for competitive ELISA; this situation produces a much smaller slope for the linear response of binding ratios to logarithmic quantities of a hapten in reaction wells and thus larger CVs due to error propagation, to account for the wider quantifiable ranges and higher LOQs of clenbuterol and penicillin G under the conditions suitable for SDESA of ALP and BGAL. Therefore, the practicability of ELISA via SDESA still

Figure 6. Application of SDESA of ALP and BGAL to ELISA of penicillin G and clenbuterol. (a) Comparison of ELISA of penicillin G via SDESA and a separate assay. (b) Comparison of ELISA of clenbuterol via SDESA and a separate assay.

requires a pair of enzyme labels with higher catalytic capacities under the conditions suitable for SDESA. 2152

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Table 3. (A) Assay of Clenbuterol by ELISA via SDESA and a Separate Assay; (B) Assay of Penicillin G by ELISA via SDESA and a Separate Assay (A) ELISA of clenbuterol via SDESA (n = 5) clenbuterol (ng/well)

penicillin G (ng/well)

1.2 30 750 750 1.2 mean SD

9 81 750 9 750

ELISA of clenbuterol via separate assay (n = 5)

B/B0 (%)

clenbuterol (ng/well)

CV (%)

recovery (%)

B/B0 (%)

clenbuterol (ng/well)

CV (%)

recovery (%)

90.8 ± 0.8 75.2 ± 4.1 60.8 ± 2.5 61.3 ± 2.9 90.6 ± 1.2

1.2 32.4 714 642 1.2

80 52 17 26 60

95 108 95 86 99 97 8

87.8 ± 1.4 74.6 ± 2.0 61.5 ± 2.2 61.8 ± 2.7 87.5 ± 1.1

1.2 29.0 730 681 1.2

63 52 49 33 53

98 97 97 91 102 97 4

(B) ELISA of penicillin G via SDESA (n = 5) clenbuterol (ng/well)

penicillin G (ng/well)

1.2 30 750 750 1.2 mean SD

9 81 750 9 750

ELISA of penicillin G via separate assay (n = 5)

B/B0 (%)

penicillin G (ng/well)

CV (%)

recovery (%)

B/B0 (%)

penicillin G (ng/well)

CV (%)

recovery (%)

87.6 ± 4.2 73.4 ± 4.3 56.7 ± 1.7 88.2 ± 2.8 57.1 ± 1.9

9.3 75 861 8.5 811

82 57 35 83 35

103 92 115 95 108 103 9

84.0 ± 1.9 69.7 ± 4.0 53.8 ± 2.0 84.5 ± 2.5 54.3 ± 1.7

9.5 78 797 8.9 736

88 47 44 66 42

105 96 106 99 98 101 5

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B as a counterpart of ALP as Enzyme A is difficult to access due to unusual optimum buffers and pH values for ALP. Hence, molecular engineering of hydrolytic enzymes acting on chromogenic substrates from 4NP to serve as Enzyme B is preferred for the practice of ELISA via SDESA for satisfactory sensitivity and ever-enhanced efficiency.

FURTHER DISCUSSION AND CONCLUSION By employing the stated criteria to select two chromogenic substrates and two wavelengths for quantifying absorbance, together with the proposed chemometrics to process data, SDESA of two enzymes of interest is generally applicable and has everenhanced efficiency besides other performances comparable to that by a separate assay under the same conditions; the application of SDESA to ELISA is effective. Hence, SDESA is promising. To practice SDESA, two enzymes in a pair should have sufficient specificity on their chromogenic substrates. In theory, cross-action of either enzyme on the chromogenic substrate of the other enzyme can be corrected by processing data with a suitable chemometrics method but surely further complicates data processing and prevents the routine practice of SDESA. As a result, for the application of SDESA to ELISA, horseradish peroxidase of low specificity on chromogenic substrates is an unsuitable label enzyme while hydrolytic enzymes bearing high group specificity for their chromogenic substrates are preferable labels for ELISA. Moreover, when there is the inactivation of either enzyme by the substrates/products of itself or the other enzyme, SDESA will be forced to process more sophisticated kinetics. In theory, a chemometrics method for such complicated situations can be worked out to mine the interference-free activities of two enzymes under SDESA, but their routine practice is surely limited. For routine practice of ELISA via SDESA, two enzymes as a pair of labels in the same reaction buffers should have sufficient catalytic capacities on their chromogenic substrates meeting the primary requirement of spectral properties. Clearly, the application of SDESA to ELISA also requires no cross-reactivity between two antibodies. Any candidate of Substrate A is perfect when λ0 is at 405 or 450 nm to facilitate the use of readily available filters on conventional microplate readers since Substrate B is easily accessed to have Product B exhibiting λB close to 405 nm (4NP) or 450 nm (4NNP).41 After intensive screening, we found the action of ALP on 4NNPP produces λ0 at 405 nm, but Enzyme

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ASSOCIATED CONTENT

* Supporting Information S

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or [email protected]. Tel: +86-23-68485240. Fax: +86-23-68485111. Author Contributions ¶

These two authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by “863″-High-Technology Program (No. 2011AA02A108), the program for New Century Excellent Talent in University (NCET-09-0928), National Natural Science Foundation of China (Nos. 30200266 and 81071427), and Natural Science Foundation Project of CQ (CSTC2011BA5039, CSTC2012JJA0081). This method is under evaluation for an invention patent in China (No. 201210355606.8) and PCT patent (PCT/CN2012/081869). 4NNPP was a gift from Chongqing Protein & Probe Biotechnology Corporation Ltd (Chongqing ProBio Co. Ltd), at Erlang, Jiulongpo Dist., Chongqing 400050, China.

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REFERENCES

(1) Allison, R. D.; Purich, D. L. Methods Enzymol. 1979, 63, 3−22.

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dx.doi.org/10.1021/ac302786p | Anal. Chem. 2013, 85, 2143−2154