Analytica Chimica Acta 854 (2015) 145–152

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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

A homogeneous assay principle for universal substrate quantification via hydrogen peroxide producing enzymes Kristin Zscharnack a , Thomas Kreisig a , Agneta A. Prasse a , Thole Zuchner a,b, * a b

Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany

H I G H L I G H T S

G R A P H I C A L A B S T R A C T

 Application of the TRF-based PATb system for universal oxidase substrate detection.  H2O2 generated by choline or glucose oxidase quenches the TRF signal of PATb.  The assay time is only limited by the oxidase catalysis rate.  Glucose is precisely detected in human serum consistent to a commercial assay.  A reliable quantification of choline in infant formula is shown.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 July 2014 Received in revised form 27 October 2014 Accepted 7 November 2014 Available online 11 November 2014

H2O2 is a widely occurring molecule which is also a byproduct of a number of enzymatic reactions. It can therefore be used to quantify the corresponding enzymatic substrates. In this study, the time-resolved fluorescence emission of a previously described complex consisting of phthalic acid and terbium (III) ions (PATb) is used for H2O2 detection. In detail, glucose oxidase and choline oxidase convert glucose and choline, respectively, to generate H2O2 which acts as a quencher for the PATb complex. The response time of the PATb complex toward H2O2 is immediate and the assay time only depends on the conversion rate of the enzymes involved. The PATb assay quantifies glucose in a linear range of 0.02–10 mmol L 1, and choline from 1.56 to 100 mmol L 1 with a detection limit of 20 mmol L 1 for glucose and 1.56 mmol L 1 for choline. Both biomolecules glucose and choline could be detected without pretreatment with good precision and reproducibility in human serum samples and infant formula, respectively. Furthermore, it is shown that the detected glucose concentrations by the PATb system agree with the results of a commercially available assay. In principle, the PATb system is a universal and versatile tool for the quantification of any substrate and enzyme reaction where H2O2 is involved. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Time-resolved fluorescence Matrix compatibility Oxidase Choline Glucose Kinetics

1. Introduction

* Corresponding author. Present address: Octapharma Biopharmaceuticals GmbH, Im Neuenheimer Feld 590, 69120 Heidelberg, Germany. Tel.: +49 3419731390; fax: +49 3419739086. E-mail address: [email protected] (T. Zuchner). http://dx.doi.org/10.1016/j.aca.2014.11.013 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.

The development of detection systems for the specific, sensitive, and low cost determination of biomolecules is important for clinical and non-clinical applications. Numerous optical read-out systems based on absorption, fluorescence, and other luminescence techniques exist. The application of time-

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resolved fluorescence as a read out method has the important advantage that samples with a high background fluorescence or absorption can be measured [1]. Fluorescence sensors are also extremely sensitive, cause little or no damage to the host system, and special fluorescence techniques can provide information about the structure and micro-environment of molecules [2]. However, most time-resolved fluorophores require complex synthesis steps and are therefore costly. We recently described a novel time-resolved fluorescence complex consisting of phtalic acid and terbium (III) ions (PATb complex), which shows a distinct time-resolved fluorescence signal and can be used in larger quantities due to the comparatively low price of the materials required [3]. As described previously, the PATb complex can be used to detect H2O2 with high specificity and sensitivity within seconds. H2O2 is also involved in a number of enzymatic reactions, for example when oxidases are used. Naturally, these enzymes convert their corresponding substrates mostly with a very high specificity. In this work, we wanted to establish a universal assay using the time-resolved PATb system for the quantification of several analytes by the conversion of their respective H2O2-producing oxidase. As examples, we chose glucose as one substrate and choline as another. Both substrates can be converted specifically by their corresponding oxidases, i.e., glucose oxidase and choline oxidase. Choline is a vitamin B-like biomolecule which is an essential nutrient for humans [4]. Humans eating low choline diets develop fatty liver and liver damage [5]. Moreover, the need for choline is increased during pregnancy due to the rapid rate of cell division and expansion of maternal and fetal tissues as well as during lactation because a high amount of choline is secreted into human breast milk [6]. Furthermore, choline is the precursor of the important neurotransmitter acetylcholine particularly necessary for memory and general intellectual ability [7]. A choline-poor diet will impair fetal brain development and increase the risk of Alzheimer’s disease [8,9]. The quantification of choline is therefore necessary for clinical samples, food industry, and pharmaceutical applications. Choline can be converted by choline oxidase (ChOx) into H2O2 and betaine. The detection of choline via the hydrogen peroxide production can be monitored by chemiluminescence [10–12], electrochemistry [13–15], and fluorescence [16,17]. In this work, we present the first choline assay based on time-resolved fluorescence with the inherent advantage of background elimination. The determination of glucose is crucial in clinical diagnosis, industrial application, and food analysis. For the diagnosis and management of diabetes mellitus, the monitoring of the blood glucose levels is very important [18]. Therefore, the development of noninvasive and continuous methods for the accurate and reliable determination of glucose is in the focus of research. For the enzymatic measurement of glucose concentrations fluorescence, colorimetric, and electrochemical methods are used [19–22]. A very popular enzyme for glucose detection is the glucose oxidase (GOx) which catalyzes the specific oxidation of glucose and therefore generates H2O2 and D-gluconolactone. The analysis of H2O2 produced during the GOx catalyzed oxidation of glucose is often monitored by fluorescence [21,23–27]. The enzymatic reaction of H2O2-generating oxidases can be measured by oxygen consumption, H2O2 production, or pH decrease [28]. As seen from the literature, there exists a multiplicity of methods for the quantification of glucose and choline holding their benefits but also a couple of drawbacks. In this work, we present the application of the H2O2 quantification via the homogeneous time-resolved PATb assay for the enzymatic determination of the model analytes choline and glucose. The innovation of the proposed PATb system consists of a combination of an easy

experimental setup, wide measuring range, short incubation time, less sample preparation, and full matrix compatibility. With both biomolecules, we show the potential of the proposed assay principle for the universal, accurate, and reproducible quantification of numerous analytes that are substrates in H2O2-producing or -converting enzyme reactions. 2. Materials and methods 2.1. Materials Terbium trichloride hexahydrate (TbCl3
6H2O), hydrogen peroxide, ortho-phthalic acid, choline oxidase (ChOx, EC 1.1.3.17, from Alcaligenes sp.), glucose, and choline chloride were purchased from Sigma–Aldrich (Steinheim, Germany). HEPES and glucose oxidase (GOx, EC 1.1.3.4, from Aspergillus niger) were ordered from VWR (Dresden, Germany). DMSO was obtained from Roth (Karlsruhe, Germany). All chemicals were of analytical grade. Deionized water was obtained from a Purelab Ultra water purification system (18.2 MV  cm). White 384 well nonbinding flat-bottom microtiter plates were purchased from Greiner Bio-One (Frickenhausen, Germany). Pooled human serum was ordered from SunnyLab (Sittingbourne, UK) or was taken from a volunteer, respectively. Infant formula was purchased from the local supermarket. For comparison with the proposed method a commercially available Abnova glucose assay kit was acquired from Acris Antibodies (Herford, Germany). 2.2. Apparatus pH measurements were performed on a Cyber scan 510 pH meter (Eutech Instruments, Netherlands) at room temperature. Dose–response curves and kinetics of the glucose and choline assay, respectively, were recorded on a Gemini EM fluorescence microplate reader (Molecular Devices, Ismaning, Germany) using bandwidths of 9 nm for both, excitation and emission. The kinetic measurement of the glucose assay with the control measurement by the addition of H2O2 or water was performed on a PerkinElmer LS45 luminescence spectrometer (Rodgau, Germany) using bandwidths of 10 nm for both, excitation and emission. Absorption of the commercially available glucose kit was recorded in a Paradigm detection platform with the SpectraMax absorbance detection cartridge (Molecular Devices, Ismaning, Germany). 2.3. Procedures 2.3.1. PATb detection solution For the 300 mmol L 1 stock solution of phthalic acid, PA was solved in DMSO. A 500 mmol L 1 stock solution of TbCl3 in water was used. Time-resolved fluorescence measurements were carried out on a Gemini EM fluorescence microplate reader (lEx = 285 nm, lEm = 545 nm, delay time = 50 ms, integration time = 1450 ms) after a 5 s shaking step at room temperature for the glucose assay or 40  C in case of the choline assay, respectively. All experiments were carried out fourfold. 2.3.2. Glucose assay GOx was dissolved in 100 mM HEPES pH 8.0 as a stock solution of 1 U mL 1, aliquoted and stored at 20  C. Glucose was solved in deionized water as a 1 mol L 1 stock solution. For the glucose assay, the following detection solution was used: 78 mL, 3 mmol L 1 PA was diluted in 100 mmol L 1 HEPES pH 8.0 and mixed with 22 mL 35 mmol L 1 TbCl3 followed by the addition of 1 U GOx per well (working concentration 1 U mL 1). For the analysis of glucose, a serial dilution of glucose (10 mL, 10 mmol L 1 to 2 mmol L 1) was

K. Zscharnack et al. / Analytica Chimica Acta 854 (2015) 145–152

mixed with the detection solution (100 mL) in a white non-binding 384 well microtiter plate. The kinetic measurement of the glucose assay was performed on a PerkinElmer LS45 luminescence spectrometer (lEx = 285 nm, lEm = 545 nm, delay time = 50 ms, integration time = 1000 ms) using a quartz cuvette 5 mm across. A volume of 100 mL PATb detection solution containing 50 mL 100 mM glucose and 1 U GOx (1 U/mL) was provided in the cuvette and 50 mL 100 mmol L 1 H2O2 was added via a capillary tube, respectively. 2.3.3. Choline assay ChOx was dissolved in 100 mM potassium phosphate buffer pH 7.0 as a stock solution of 1 U mL 1, aliquoted and stored at 20  C. Choline chloride was solved in deionized water as a 100 mmol L 1 stock solution. For the choline assay a concentrated detection solution was used as follows: 15 mL, 14 mmol L 1 PA was diluted in 100 mmol L 1 HEPES pH 8.5 and mixed with 10 mL 70 mmol L 1 TbCl3 followed by the addition of 0.5 U ChOx per well (working concentration 0.02 U mL 1). For the choline determination, a serial dilution of choline chloride (50 mL, 200 mmol L 1 to 0.4 mmol L 1) was mixed with 50 mL concentrated detection solution in a white non-binding 384 well microtiter plate. The preparation was incubated 20 min at 40  C in the Gemini fluorescence reader and was measured as stated above (see Section 2.3.1). 2.3.4. Measurement in real samples The detection solutions were the same as described above for the glucose and choline assay, respectively. Serum and formula were diluted 1:100 in water and were spiked with a standard glucose or choline chloride solution, respectively. Each concentration was analyzed four times in parallel.

147

Glucose and choline were chosen as model biomolecules to demonstrate the universal applicability of the PATb system to determine analytes via H2O2 production of their corresponding oxidases. In the first model assay described here, glucose oxidase (GOx) converts glucose and oxygen into H2O2 and D-gluconolactone. The quantification of H2O2 which is directly proportional to glucose concentrations is performed by the measurement of the timeresolved fluorescence using the PATb assay. The emission signal is decreasing over time as well as with increasing glucose concentrations as H2O2 quenches the emission of the PATb complex. A serial dilution of glucose was incubated with GOx for 30 min and as expected, the decrease of the fluorescence intensity at 545 nm was detected over time (Fig. 1A). Due to the accumulation of H2O2 amounts, the precision of the glucose assay is increasing and the linear range as well as the LOD shifts to lower glucose concentrations. An enzyme reaction time of 10 min was determined to be optimal for a suitable detection of glucose with the proposed assay and resulted in the precise assay parameters described below. In Fig. 1B the fast response of the PATb complex toward H2O2 is demonstrated. The signal–response curve of the glucose assay containing the substrate glucose, GOx, and the PATb complex is monitored over time. When the signal becomes nearly stable, 100 mmol L 1 H2O2 or the same volume of water as a control was added. The response of the time-resolved fluorescent PATb complex to the quenching of H2O2 as expected by the underlying collisional quenching mechanism is immediate and seems to be only limited by mixing and dilution effects. To the best of our knowledge, the PATb assay is the fastest system described so far for the detection of

2.3.5. Assay quality control Intra-assay variation was calculated from the results of four parallel experiments, inter-assay variation was determined from the mean of the calculated concentrations of three independent assays. Analytical recovery was calculated as the quotient of measured and known concentrations from four analyte concentrations using a serial dilution of glucose or choline chloride in water in a concentration range as indicated in the respective calibration plots. All samples were analyzed four times in parallel. 2.3.6. Commercial glucose assay kit The measurements were performed according to the manufacturer’s manual. Therefore, human serum samples were diluted 1:100 in the glucose assay buffer and spiked with a standard glucose solution. Absorbance at 570 nm was read on a Paradigm detection platform (E = 570 nm). All samples were analyzed as triplicates. 2.3.7. Statistical analysis The limit of detection was defined as meanblank – 3  SDblank. The measured values below this calculated limit were interpreted as positive signals. Values of dose–response curves were subjected to a Dixon and Hampel outlier test [29,30]. Outliers identified by both tests (p = 0.95) were eliminated. 3. Results and discussion 3.1. Optimization of the glucose assay Recently, we described the PATb assay for the sensitive, reliable, and fast detection of H2O2 based on time-resolved fluorescence [3]. A complex consisting of phthalic acid and terbium ions is excited at a wavelength of 285 nm and the emission at 545 nm is quenched by collisional quenching dependent on the H2O2 concentration.

Fig. 1. Kinetic measurement of the glucose assay. (A) Different concentrations of glucose (0–2.5 mmol L 1) were incubated with 1 U GOx and the PATb complex at room temperature. (B) 5 nmol glucose were converted by 1 U GOx over time and H2O2 production was monitored by the PATb complex (dotted line). After 950 s, 50 mL of a 100 mmol L 1 H2O2 solution or the same volume of water as a control was added as indicated by an arrow and an immediate response of the PATb system to H2O2 was observed.

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Glucose oxidase (U)

Linear range (mmol L

0.01 0.1 1

0.78–25 0.16–5 0.02–1.25

a

LOD, limit of detection (meanblank

1

)

LOD (mmol L

1 a

)

0.78 0.04 0.002

3  SDblank).

H2O2 based on time-resolved fluorescence. The assay time for the determination of the model analyte glucose only depends on the reaction time of GOx. In human serum samples the reference range for glucose is 3.9–7.2 mmol L 1 and is increased up to 16.5 mmol L 1 in case of diabetes [31]. Considering the dilution of the serum samples to receive a compatibility with the glucose assay, glucose concentrations should be detected in the lower mmol L 1 range. Therefore, the optimal GOx amount was investigated by recording a signal–response curve for a glucose dilution series incubated with 0.01, 0.1, and 1 U GOx, respectively. After a reaction time of 10 min the linear ranges and the detection limits (LOD, meanblank – 3  SDblank) were determined and are summarized in Table 1. The linear ranges as well as the LOD shift with higher amounts of GOx to lower glucose concentrations. Larger amounts of GOx led to a faster conversion of glucose into H2O2 which resulted in a lower detection range of glucose concentrations within a 10 min reaction time. The use of 1 U GOx was considered as optimum considered in terms of the linear range and LOD for the determination of glucose in serum samples. Glucose assays based on electrochemiluminescence require often complex, time consuming, and therefore costly synthesis steps to generate the respective sensors. In terms of linear range and detection limit these sensors are partially more sensitive or have a wider measurement range compared to our assay (Table 6) [17,32,33]. The proposed glucose assay based on the PATb complex combines the benefit of an easy experimental setup with a wide linear range, a LOD as good as the published methods (Table 6), and additionally a short incubation time. Furthermore, the PATb based glucose assay has the advantage of background reduction due to the time-resolved measurement. In comparison to published fluorescence based glucose assays our systems is characterized with a broad measuring range and simultaneously low detection limits (Table 6). Most other methods need three times higher incubation times and higher incubation temperatures [34,35]. To investigate the precision and reliability of the glucose assay the intra- and inter-assay variances were determined with 1 U GOx after 10 min incubation time (Table 2). The average intra-assay variance is 3.13%, the average inter-assay variance is 7.08 % (Table 2). These data demonstrate the good reproducibility of the

Table 2 Intra- and inter-assay variances of glucose concentrations were determined by a calibration plot with 1 U GOx after a 10 min incubation step. Glucose standard (mmol L 1)

0.03 0.06 0.12 0.25 0.5 1 a

Coefficient of variance.

Glucose found Intra-assay (n = 4)

Inter-assay (n = 3)

mean  SD (mmol L 1)

CV (%)a

mean  SD (mmol L 1)

2.95 4.59 5.00 1.33 3.23 1.68

0.034 0.054 0.108 0.243 0.51 0.94

0.035 0.059 0.1345 0.2806 0.497 1.069

     

0.001 0.003 0.007 0.004 0.016 0.018

     

glucose detection with the glucose assay based on the terbium complex PATb. 3.2. Glucose determination in human serum samples For the measurement of glucose levels in human serum, the serum compatibility of the glucose assay was investigated. Human serum was diluted in water to receive a final concentration of 1%, 2.5%, 5%, and 10% serum, respectively. A serial dilution of glucose was prepared in the corresponding serum solutions. A good overlay of the signal curves in serum with the calibration plot in water was obtained up to a serum concentration of 5%. Therefore, the determination of glucose levels is possible until a maximum serum concentration of 5% in the assay preparation. For best precision of the glucose assay, the serum samples were diluted 100 fold. No other pretreatments like deproteinization and centrifugation typically required for the analysis of biological samples were necessary. Due to the high sensitivity of the PATb probe, the quantifiable glucose concentrations in the diluted samples lie within the reference range for glucose levels in human serum. Recoveries were determined in serum spiked with defined glucose concentrations. Glucose levels were measured with the PATb assay using the calibration plot with a serial dilution of glucose concentrations in water (Fig. 2). Recoveries are found between 102.4% and 109.7% with a relative standard deviation of an average of 6.4% (Table 3). These values indicate the good accuracy and precision of the glucose assay not only in buffer but also in serum samples. 3.3. Correlation of the PATb based glucose assay to a commercially available assay To estimate the precision of the glucose assay, we compared the PATb assay with a conventional glucose assay kit based on a colorimetric measurement of H2O2. Therefore, human serum was diluted in water or the appropriate buffer, respectively, and spiked with defined glucose concentrations. The proposed glucose assay plotted on the ordinate showed a good correlation (y = 0.9885x; R2 = 0.976) with a commercial glucose assay kit represented on the abscissa (Fig. 3). The PATb based glucose assay provides a precise and reliable determination of glucose in human serum samples in 10 min compared to the 30 min incubation time for the commercially available assay. The 300 Fluorescence intensity (×106)

Table 1 Glucose assay parameters in dependence of GOx amount after a 10 min incubation step.

250 200 150 100 50

CV (%)a

0.0004 1.21 0.005 5.91 0.019 17.47 0.031 10.74 0.027 4.63 0.161 2.49

0 0.001

0.01

0.1

1

10

Glucose (mmol L-1) Fig. 2. Signal–response curve (semi logarithmic plot) of the glucose assay measured with 1 U GOx after a 10 min incubation step. The dashed line indicates the limit value for the LOD (meanblank 3  SDblank; 2 mmol L 1). The linear range (0.02–1.25 mmol L 1) which was used as calibration plot is indicated by the solid regression line (y = 5.29  107ln(x) + 4.07  107; R2 = 0.9903).

K. Zscharnack et al. / Analytica Chimica Acta 854 (2015) 145–152 Table 3 Recoveries of the glucose assay found in spiked human serum samples.

0 0.03 0.06 0.12 1 Average

a

1

)

Found (mmol L

1

)

0.053 0.082 0.132 0.177 1.138 –

CV (%)a

Recovery (%)

9.9 5.3 9.3 5.0 2.3 6.4

– 102.4 109.7 103.5 108.0 105.9

Coefficient of variance, n = 4.

precision and the LOD of the proposed method are better in comparison to the commercially available assay. The results indicate that the PATb based glucose assay provides an alternative method for the determination of glucose in human serum samples with the advantages of background reduction, wide linear range, ten times better detection limit, and a faster response time. 3.4. Optimization of the choline assay To demonstrate the wide applicability of the PATb assay to quantify different substrates, a second application via the use of choline oxidase (ChOx) was tested. As a second model system for the application of the time-resolved fluorescence PATb system we chose choline for the determination in infant formula. The ChOx catalyzes the oxidation of choline to form betaine and H2O2. With the PATb assay, the production of H2O2 was detected which is directly proportional to the choline concentration. The emission signal of the PATb complex is decreasing over time as well as with increasing choline concentrations as H2O2 quenches the emission of the PATb complex. Signal–response curves of the choline assay were measured for a dilution series of choline chloride over time (Fig. 4) and showed a choline concentration dependent signal development. A sensitive and quantifiable signal decrease could be observed in dependence of the choline concentration. Interestingly, even in the absence of choline a decrease of the fluorescence intensity could be observed. This is most likely caused by an unspecific effect due to the relatively high ChOx protein concentration, but did not affect the assay parameters negatively as described below. For the determination of the linear range of the PATb choline assay, a calibration plot was measured from a dilution series of choline chloride in the presence of 0.5 U ChOx at 40  C (Fig. 5). After an incubation time of 20 min, linearity of the measured curve between 1.56 and 100 mmol L 1 choline chloride could be observed

PATb assay (mmol L-1)

0.25

0.2 0.15 0.1 y = 0.9885x 0.05

Fluorescence intensity (×106)

Added (mmol L

120

choline chloride mmol L-1 0

100

1.56 3.13

80

6.25 12.5

60

25 50

40 20 0 0

500

Fig. 3. Correlation of the PATb glucose assay and a commercially available glucose assay kit from Abnova. Human serum samples were diluted 100 fold in water (PATb) or buffer (kit) and spiked afterwards with defined amounts of glucose.

2000

40 35 30

25 20

15 10

5 0 0.1

1

10 100 choline chloride (µmol L-1)

1000

Fig. 5. Signal–response curve (semi logarithmic plot) of the choline assay measured with 0.5 U ChOx after a 20 min incubation step at 40  C. The dashed line indicates the limit value for the LOD (meanblank 3  SDblank; 1.56 mmol L 1). The linear range (1.56–100 mmol L 1) which was used as calibration plot is indicated by the solid regression line (y = 8.54  106ln(x) + 4.33  107; R2 = 0.9919).

and the detection limit was 1.56 mmol L 1. To the best of our knowledge, this choline assay is the first one based on timeresolved fluorescence with the inherent advantage of a reduced background signal originating from the microtiter plate material or from the sample matrix. The above mentioned assay parameters like linear range, LOD, and incubation time are comparable to published choline assays based on fluorescence [16,17], chemiluminescence [12], and electrochemiluminescence [10]. The intra- and inter-assay variances of the choline assay were investigated using a standard choline chloride solution (Table 4). Table 4 Inter- and intra-assay variances of choline concentrations were determined by a calibration plot in water with 0.5 U ChOx after a 20 min incubation step at 40  C.

0 0.1 0.2 0.3 abnova glucose assay kit (mmol L-1)

1500

45

Choline standard (mmol L

0

1000 Time (s)

Fig. 4. Kinetic measurement of the choline assay. Different concentrations of choline chloride (0–50 mmol L 1) were incubated with 0.5 U ChOx and the PATb complex at 40  C.

Fluorescence intensity (× 106)

Samples 1 2 3 4 5

149

5 10 15 30

1

)

Choline found Inter-assay (n = 3)

Intra-assay (n = 4)

Mean  SD (mmol L 1)

CV (%)

Mean  SD (mmol L 1)

2.40 16.00 9.80 10.56

4.36 8.81 15.30 34.89

4.43 9.93 16.44 37.71

   

0.11 1.59 1.61 3.99

   

0.21 0.73 0.83 0.63

CV (%) 4.73 8.33 5.42 1.81

150

K. Zscharnack et al. / Analytica Chimica Acta 854 (2015) 145–152 Table 5 Recoveries of the choline assay found in spiked infant formula samples.

Fluorescence intensity (× 106)

35

Samples

Added (mmol L

20

1 2 3 4

5 10 15 30 Average

15

a

30 25

1

)

Found (mmol L

1

)

5.54 10.43 13.66 30.18 –

CV (%)a

Recovery (%)

3.4 7.3 3.1 0.5 3.6

110.7 104.3 90.9 100.6 101.6

Coefficient of variance, n = 4.

10 water 1% infant formula

5 0 0.1

1

10

100

1000

choline chloride (µmol L-1) Fig. 6. Compatibility of the choline assay to infant formula (semi logarithmic plot). Signal–response curves of a serial dilution of choline in formula (1%) in comparison to water are shown with 0.5 U ChOx after a 30 min incubation step at 40  C. The regression line (y = 5.75  106ln(x) + 3.1 107; R2 = 0.9915) is given for the linear range measured in water.

The average intra-assay variance is 5.07%, the average inter-assay variance is 9.69% (Table 4). These data represent the satisfactory reproducibility of the choline detection with the choline assay based on the terbium complex PATb and confirm the high reproducibility of the PATb system when used for the detection of choline, besides glucose as the first model system tested in this study. 3.5. Determination of choline in infant formula As formula is the single nutrition source for non breast-fed infants it is of outstanding importance to precisely quantify the ingredients. Choline is added as choline chloride to infant formula at a minimum level of 7 mg/100 kcal to a guidance upper level of 150 mg/100 kcal [36], which corresponds to a choline concentration of 0.3 mmol L 1 to 7.2 mmol L 1.

Fig. 6 shows the compatibility of the choline assay to infant formula. Therefore, signal–response curves of a serial dilution of choline chloride were prepared in water and 1% infant formula, respectively. An excellent overlay of the curve obtained for choline concentrations in 1% formula to the calibration plot in water was observed within the linear range. For the determination of the recoveries, so called follow-up milk was selected since it contains no choline while the composition of the other ingredients is essentially the same compared to the initial formula. Recoveries were determined in 1% follow-up formula spiked with defined choline concentrations. Choline levels were measured with the PATb assay using the calibration plot with a serial dilution of choline concentrations in water (Fig. 6). Recoveries were between 90.9% and 110.7% with an average RSD of 3.6% (Table 5). These values demonstrate the good accuracy and precision of the choline assay. The excellent assay parameters observed for the determination of choline and glucose via the PATb assay system show, that the inexpensive PATb system is suitable for the quantification of a wide range of enzyme substrates when H2O2 is involved in the enzyme reaction. Interestingly, the immediate signal generation in principle also allows a determination of enzyme kinetics. At the same time, this work presents the first assays for the quantification of choline based on time-resolved fluorescence. Most other assays are based on electrochemiluminescence with the assay parameters summarized in Table 6. Compared to the literature, the oxidasebased PATb assay has a wide linear range with detection limits in the same range. The response time of H2O2 detection by the PATb

Table 6 Comparison of different methods for glucose and choline determination based on the detection of H2O2. Detection

Nanomaterials

Glucose assay (GOx) Linear range (mmol L

Fluorescence Electrochemiluminescence Electrochemiluminescence Chemilumi-nescence Electrochemiluminescence Electrochemistry Fluorescence Fluorescence Time-resolved Fluorescence Time-resolved Fluorescence Time-resolved Fluorescence Fluorescence Fluorescence Chemiluminescence Time-resolved Fluorescence -, Not specified.

1

LOD (mmol L

Choline assay (ChOx) Incubation 1 ) time

Refs.

Linear range (mmol L 1)

LOD (mmol L

1

)

Incubation time

)

Molecular beacon IDA/Sepharose/PVA-SbQ chip; luminol

10–250 20–2000

2 20

60 min 3 min

5–100 2–200

1 2

60 min 3 min

[37] [38]

GOx/MWCNTs/CS/ITO electrodes

20–2000

14

-

100–1000

97

-

[39]

Graphene oxide (GO)/lucigenin nanocomposite PBBIns-Gs/Au electrode

5.6– 27,800 10–10,000

5.5

50 min

41–4100

62

50 min

[33]

5.6 s

0.1–830

0.02

-

[40]

Os-gel-HRP/GOx Graphene oxide-DNA sensor PVA-pyrene Mn-doped ZnS quantum dots

0.1–30 0.5–10 250–3000 10–100

0.05 0.1 190 3

20 min 0.1–20 3 h 30 min 15 min 30 min; 40  C

0.05

20 min

[32] [41] [42] [34]

Eu3Tc

2.7–100

2.2

Monolithic silica gel, GOx

400–5000

170

30 min; 30  C -

C-dots, fenton reaction CdTe quantum dots ZnO nanorod films PATb

20–1250

–

2

10 min

[35] [43] 0.1–40 5–150 6–2000 1.56–100

0.1 0.5 1.56

30 min 10 min 20 min

[16] [17] [12] This work

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system is immediate. The incubation time for the GOx- or ChOxcoupled PATb assay only depends on the reaction rate of the enzymes and is faster than most of the comparable assays (Table 6). The PATb system is a homogeneous assay where timeconsuming and expensive assembly of a sensor, washing steps or several working steps are not required. The time-resolved measurement of the fluorescence signal allows a backgroundindependent determination in different sample matrices like serum and infant formula as described above. 4. Conclusion In this work, we introduce the PATb complex [44] as a universal assay principle for the analyte quantification due to the H2O2 detection via their corresponding oxidase reaction. For the exemplified applicability, the quantification of two important biomolecules, glucose and choline, was shown successfully. By the production of H2O2 the emission signal of the complex formed by phthalic acid and terbium ions is quenched. The timedependent decrease of the fluorescence signal is directly proportional to the analyte amount. With the PATb assay glucose and choline were determined precisely, reproducible, and reliable. The rate of the reaction is limited to the speed of the catalysis of the oxidases. As the analytes were measured in human serum and infant formula, the PATb assay shows a wide applicability to real samples with sufficient precision and reproducibility. An advantage of the proposed method is the measurement of the fluorescence signals in a time-resolved mode since the background from complex matrices is eliminated. The PATb assay is easily adaptable for the quantification of further biomolecules by the use of other H2O2-generating oxidases (e.g., alcohol oxidase, NADPH oxidase, lactate oxidase) and other H2O2converting enzymes (e.g., catalase, myeloperoxidase, glutathione peroxidase). Acknowledgements We gratefully acknowledge the expert technical assistance from Stefanie Langanke. The authors thank Prof. Ralf Hoffmann for lab access and helpful discussions as well as Kristin Dobslaff for proof-reading. This work was supported by the Federal Ministry of Education and Research (BMBF) GO-Bio project no. 0315988 to TZ.

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