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A sensitive one-step method for quantitative detection of α-amylase in serum and urine using a personal glucose meter† Qing Wang, Hui Wang, Xiaohai Yang,* Kemin Wang,* Rongjuan Liu, Qing Li and Jinqing Ou Assays of α-amylase (AMS) activity in serum and urine constitute the important indicator for the diagnosis of acute pancreatitis, mumps, renal disease and abdominal disorders. Since these diseases confer a heavy financial burden on the health care system, AMS detection in point-of-care is fundamental. Here, a onestep assay for direct determination of the AMS activity was developed using a portable personal glucose meter (PGM). In this assay, maltopentaose was used as a substrate for sensitive detection of AMS with the assistance of α-glucosidase. In the presence of AMS, maltopentaose can be readily hydrolyzed to form maltotriose and maltose quickly. With the enzymatic hydrolysis of α-glucosidase, maltotriose and maltose can be turned into glucose rapidly, which can be quantitatively measured using a portable PGM. This assay did not require any cumbersome and time consuming operations, such as surface modification, synthesis of invertase conjugate, washing and centrifugation. Detection of AMS can be achieved using only a one-step mixture, and the limit of detection was 20 U L−1 which was lower than the clinical cutoff for AMS. More importantly, this sensitive and selective assay was also used for the detection of AMS in

Received 4th November 2014, Accepted 4th December 2014

human serum/urine samples. The results showed that the recovery of AMS from human serum/urine

DOI: 10.1039/c4an02033b

samples was 91–107%. The rapid and easy-to-operate assay may have potential application in the fields of point-of-care (POC) clinical diagnosis, particularly in rural and remote areas where lab equipment

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may be limited.

Introduction α-Amylase (AMS) is an endoglycosidase that catalyzes the hydrolysis of 1,4-α-glucosidic linkages between adjacent glucose units in complex carbohydrates, such as starch and glycogen, yielding glucose and maltose. The concentration of AMS in serum is normally low and fairly constant. It can increase within a few hours because of the onset of acute pancreatitis and salivary gland inflammation.1–3 Determination of AMS in serum and urine is mainly requested to help in the diagnosis of acute pancreatitis, mumps, renal disease and abdominal disorders.3 The amyloclastic4 and the saccharogenic5 methods are the most traditional procedures for AMS detection. The former is based on the starch–iodine reaction, and the latter is based on the formation of reducing sugars,

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China. E-mail: [email protected], [email protected]; Fax: +86 731 88821566; Tel: +86 731 88821566 † Electronic supplementary information (ESI) available: The effect of maltopentaose concentration. See DOI: 10.1039/c4an02033b

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measured as maltose or glucose equivalents. However, these methods are time consuming, and the endogenous glucose interference and the unstable reaction colors result in poor reproducibility and reliability. Later, other methods were developed, such as the fluorescence method,6–8 immunological methods,9,10 flow injection spectro-photometric analysis,11 Fourier transform infrared spectroscopy,12 asymmetric split-ring resonator-based biosensor13 and electrochemical sensors.14,15 Most of these methods showed high sensitivity, but a series of reagents, relatively expensive equipment and trained operators were required, which is limited in a remote area or at home. Therefore, there is an unmet need for developing simple methods to detect AMS at the point of care (POC). A personal glucose meter (PGM) is one of the successful portable devices for POC testing, due to its compact size, low cost, reliable quantitative results and simple operation. Generally, PGM is only used for the detection of glucose.16 Recently, it was reported that PGM can be used to detect other targets with the assistance of invertase conjugate, including metal ions,17,18 small molecules,19,20 nucleic acids,21 proteins21–24 and pathogenic bacteria.25 Even multiple-target detection was achieved using PGM in combination with microfluidic technology in our recent work.26 Most of these studies still need

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37 °C. Then different concentrations of AMS were added. After incubating for 15 min at 37 °C, the above reaction solutions were detected using a personal glucose meter (PGM). The PGM with a dynamic range of 2.2–27.8 mM is a product of Sinocare Inc. (China). For identifying the target-specificity of this assay, four proteins, i.e. HSA, hemoglobin, CRP and thrombin, were respectively reacted with the mixture of maltopentaose and α-glucosidase. After incubating for 15 min at 37 °C, the reaction solutions were detected using the PGM. Fig. 1

The strategy for quantifying α-amylase using PGM.

Detection of AMS in serum/urine samples some cumbersome operations, such as the syntheses of invertase conjugate, surface modification and washing steps. Hemakesh et al. introduced a simple assay for quantifying active enzyme analytes using PGM.27 Though neither surface modification nor the washing step was required in this simple assay, some specific substrates need to be designed and synthesized. Here, a one-step sensitive assay for quantitative detection of AMS was developed using PGM as the readout. As illustrated in Fig. 1, maltopentaose was used as the substrate for sensitive detection of AMS by coupling with α-glucosidase. When AMS was present, it reacted with maltopentaose to release maltotriose and maltose. Then α-glucosidase catalyzed hydrolysis of maltotriose and maltose into glucose subsequently, which was quantitatively monitored using a portable PGM. Unlike the previous studies using PGM,17–19,21–26 the cumbersome and time consuming operations, such as surface modification, synthesis of invertase conjugates, washing and centrifugation, are not required. In this assay, the user only deposits a sample into a pre-loaded tube containing maltopentaose and α-glucosidase, waits for some minutes, and then measures the quantity of glucose in the reaction solution. This rapid, sensitive and easy-to-operate assay may hold great promise for further applications in the fields of POC clinical diagnosis, particularly at home or remote areas where the laboratory services are limited.

Experimental Materials and reagents α-Amylase (AMS) and α-glucosidase were purchased from Sigma-Aldrich Corp. (USA). C-reactive protein (CRP) and hemoglobin were purchased from Biovision (USA). Thrombin and human serum albumin (HSA) were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd (China). Maltopentaose was purchased from J&K Scientific Ltd (China). All the chemical reagents were of analytical grade or higher. Ultrapure water (18.2 MΩ cm) was used throughout. Procedures of AMS detection in buffer solution Maltopentaose (30 mM) and α-glucosidase (2000 U L−1) were first mixed in a PBS buffer (10 mM, pH = 7.3) for 15 min at

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Pretreatment of the human serum sample and urine sample. The serum sample and urine sample from healthy individuals were obtained from a hospital. The samples were first centrifuged at 1000g for 5 min. The supernatants were then stored for further use. Since the activity of AMS can be influenced at low pH values, the pH of urine samples should be adjusted to 7.0 before use. The AMS-spiked serum/urine samples were prepared by adding AMS to the above serum/ urine samples. Standard curve. 40 µL Serum/urine samples, 5 µL maltopentaose (final concentration: 30 mM) and 5 µL α-glucosidase (final concentration: 2000 U L−1) were incubated at 37 °C for 5 min. Then different concentrations of AMS were added. After incubating at 37 °C for 15 min, the reaction solution was monitored using the PGM. Next the standard curve was drawn by plotting AMS concentration on X-axis and enhanced PGM signal on Y-axis. AMS detection. Since there already exists a detectable amount of glucose and AMS in the human serum and urine sample, this original glucose and AMS concentration has to be taken into account for the accurate quantification of AMS. The glucose in the serum/urine samples was first detected using the PGM and recorded as G0glu. Secondly, maltopentaose (final concentration: 30 mM) and α-glucosidase (final concentration: 2000 U L−1) were mixed in the serum/urine sample. After incubating for 15 min at 37 °C, the reaction solution was detected and recorded as G0AMS. This value demonstrated the sum of the amount of the original glucose in the unspiked serum/urine sample and that of glucose which was produced by the original AMS in the unspiked serum/urine sample. Based on the difference between G0AMS and G0glu, the AMS concentration in the unspiked sample (A0) was calculated using the standard curve. Thirdly, the AMS spiked serum/urine samples were incubated with the mixture of maltopentaose (final concentration: 30 mM) and α-glucosidase (final concentration: 2000 U L−1) at 37 °C. After 15 min, the reaction solution was monitored and recorded as GAMS. This value demonstrated the total amount of glucose determined in the AMS-spiked serum/urine samples. According to the above standard curve and the difference between GAMS and G0glu, the AMS concentration in the spiked samples (A′) was obtained, resulting in calculating the recovery of AMS subsequently.

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Results and discussion

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Optimization of the experimental conditions The sensitivity of the assay originated from the enzyme activity. Since the temperature affected the activity of enzyme, the effect of temperature was first investigated. As shown in Fig. 2, the signal of PGM increased obviously as the temperature increased in the range of 10–37 °C. As the temperature increased over 37 °C, the signal of the PGM decreased obviously. The results imply that both low temperature and high temperature may decrease the enzyme activity. Hence, 37 °C was selected as the optimum reaction temperature. To obtain the optimum sensitivity, the effect of the concentration of maltopentaose and the catalytic reaction time were also investigated. As shown in Fig. 3, when the maltopentaose concentration was stable, the signal of the PGM increased obviously as the catalytic reaction time increased. Similarly, when the catalytic reaction time was kept constant, the signal

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of the PGM increased along with the increase of maltopentaose concentration, and then became saturated at 30 mM (shown in Fig. 3 and Fig. S1 of ESI†). In addition, as the maltopentaose concentration was low (for example: 10 mM), only a long reaction time can result in a detectable signal (Fig. S1A of ESI†). The obvious signal was achieved within a short reaction time as the maltopentaose concentration was higher than 20 mM (Fig. S1B–S1C†). Moreover, as shown in Fig. 3, different concentrations of AMS could be differentiated significantly at 15 min of catalytic reaction time. Hence, a maltopentaose concentration of 30 mM and a reaction time of 15 min were used in the following experiment. Detection of AMS in buffer solution Different concentrations of AMS were detected under the following experimental conditions: reaction time, maltopentaose concentration and incubation temperature were 15 min, 30 mM and 37 °C, respectively. As shown in Fig. 4A, the PGM reading gradually increased as the AMS concentration

Fig. 2 The effect of temperature. Reaction time and maltopentaose concentration were 15 min and 30 mM, respectively. The error bars represent the standard deviation of three measurements. A signal of PGM larger than 2.2 mM was regarded as signal.

Fig. 3 The effect of reaction time. The incubation temperature was 37 °C. The error bars represent the standard deviation of three measurements. A signal of PGM larger than 2.2 mM was regarded as signal.

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Fig. 4 The sensitivity (A) and selectivity (B) of AMS detection. Reaction time, maltopentaose concentration and incubation temperature were 15 min, 30 mM and 37 °C, respectively. A signal of PGM larger than 2.2 mM was regarded as the signal.

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increased from 0 to 1000 U L−1. The calibration equation is y = 2.842 + 0.01276x (R2 = 0.9896) for AMS in buffer. Here, y is the PGM signal and x is the concentration of AMS. According to the 3σ rule, the signal could be identified even at an AMS concentration as low as 20 U L−1, which was comparable to or better than that of those in previous studies.6,8–10,12 At normal levels, AMS is present in serum below 220 U L−1 and present in urine below 800 U L−1, whereas the concentration is greater than 220 U L−1 in serum or 800 U L−1 in urine as in most of patients with acute pancreatitis. The detection limit of the assay shown in Fig. 4A was lower than the clinical cutoff for AMS, suggesting that the assay was sufficient for differentiating individuals with normal and acute pancreatitis patients. Besides sensitivity, the selectivity of this approach was also investigated. Four proteins, i.e. thrombin, hemoglobin, HSA and CRP, were used as contrasts. As shown in Fig. 4B, the presence of AMS led to remarkable increase of the PGM signal, while almost no signal could be measured in the presence of the other four proteins. It implied that this assay showed excellent selectivity for AMS detection. The result might be due to the specificity of enzyme.

Detection of AMS in real samples Given the high sensitivity and excellent selectivity of this assay, we also used it to detect AMS in the human serum and urine sample. The AMS-spiked serum/urine samples were first prepared by adding AMS to the human serum/urine samples. Next, standard curves of AMS in serum/urine were recorded. As shown in Fig. 5, for AMS in serum/urine, the PGM signal gradually increased as the AMS concentration increased from 0 to 1000 U L−1, and the linear relationship between AMS concentration and PGM signal was also established. The calibration equations are Δy = 0.191 + 0.01339x (R2 = 0.9975) for AMS in serum (Fig. 5A) and Δy = −0.153 + 0.01491x (R2 = 0.9967) for AMS in urine (Fig. 5B), respectively. Here, x is the concentration of AMS and Δy is the enhanced PGM signal which is obtained by subtracting the original glucose in serum/urine samples (i.e. G0glu) from the final measured signal (i.e. GAMS). Then, six AMS spiked serum/urine samples were measured and the AMS concentrations were checked based on the above standard curve. Next, the recovery of AMS in serum/urine was calculated and is listed in Table 1. Here, A0 is the AMS concentration determined in the unspiked sample. ΔA is the spiked concentration of AMS. A′ indicated the AMS concentration determined in the spiked sample. As shown in Table 1, the recovery of AMS from human serum/urine samples was 91–107%. The results demonstrated that the other components of the serum and urine did not interfere significantly in this assay. Thus the assay implied its potential for practical applications in disease diagnostics. Generally, if serum amylase is normal, but acute pancreatitis is strongly suspected, urine amylase may be measured to identify a delayed presentation. Given that the concentration of AMS is greater than 800 U L−1 in the urine of acute pancrea-

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Fig. 5 The standard curve of AMS in human serum (A) and urine (B). The enhanced PGM signal was obtained by subtracting the original glucose in serum/urine samples from the final measured signal. The error bars represent the standard deviation of three measurements.

Table 1

Detection of AMS in human serum and urine samplesa

Blood sample 1 Blood sample 2 Blood sample 3 Urine sample 1 Urine sample 2 Urine sample 3

A0 (U L−1)

ΔA (U L−1)

A′ (U L−1)

Recovery

66.7 66.7 66.7 125.0 125.0 125.0

500 200 100 800 500 100

598.1 262.1 157.5 915.7 667.5 231.5

106% 98% 91% 99% 109% 107%

A0, AMS concentration determined in the unspiked sample; ΔA, spiked concentration of AMS; A′, AMS concentration determined in the spiked sample. Recovery = (A′ − A0)/ΔA.

a

titis patients, we reasoned that shorter assay time could meet the urine test for AMS. As shown in Fig. 6, as the reaction time was longer than 3 min, a detectable signal could result for high concentration of AMS (>800 U L−1). When the reaction time was longer than 4 min, this assay can differentiate between low concentration of AMS (800 U L−1), implying that it can differentiate normal individuals and most patients with acute pancreatitis through a 4 min assay for urine test. Given these promising results, we reasoned that

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Fig. 6 Detection of high concentration of AMS at short time. The incubation temperature was 37 °C. Concentration of maltopentaose was 30 mM. The error bars represent the standard deviation of three measurements. A signal of PGM larger than 2.2 mM was regarded as the signal.

shorter assay times would further improve the convenience of the assay for POC application.

Conclusions In summary, a novel and simple assay for measuring the AMS activity was developed using PGM as readout, without any cumbersome and time consuming operation. The sensitive and selective assay could be implemented rapidly and easily, even in complex samples such as serum and urine, thus making it amenable for use in POC application.

Acknowledgements This work was supported by the National Natural Science Foundation of China (21190040, 21375034, 21175035), National Basic Research Program (2011CB911002), International Science & Technology Cooperation Program of China (2010DFB30300), the Fundamental Research Funds for the Central Universities and the China Scholarship council (201308430175).

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A sensitive one-step method for quantitative detection of α-amylase in serum and urine using a personal glucose meter.

Assays of α-amylase (AMS) activity in serum and urine constitute the important indicator for the diagnosis of acute pancreatitis, mumps, renal disease...
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