Point-of-Care Detection of β-Lactamase in Milk with a Universal

Letter pubs.acs.org/ac

Point-of-Care Detection of β‑Lactamase in Milk with a Universal Fluorogenic Probe Yiping Chen,†,§ Yunlei Xianyu,†,§ Jing Wu,† Wenfu Zheng,*,† Jianghong Rao,*,‡ and Xingyu Jiang*,† †

Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, 11 BeiYiTiao, ZhongGuanCun, Beijing 100190, China ‡ Molecular Imaging Program at Stanford, Departments of Radiology and Chemistry, Stanford University, 1201 Welch Road, Stanford, California 94305-5484,United States S Supporting Information *

ABSTRACT: The illegal addition of β-lactamase (Bla) in milk to disguise β-lactam antibiotics has been a serious issue in the milk industry worldwide. Herein, we report a method for pointof-care detection of Bla based on a probe, Tokyo Green-tethered β-lactam (CDG-1), as a common substrate of various Blas (Bla A, B...) which can enzymatically convert CDG-1 (low fluorescence) to Tokyo Green (high fluorescence). This approach allows rapid screening of a broad spectrum of Blas in real milk samples within 15 min without any pretreatment. Combined with the immuno-magnetic separation, we achieved sensitive and quantitative detection of Bla (10−5 U/mL), which provides a universal platform for screening and determining Blas in complex samples with high efficiency and accuracy. he β-lactam antibiotics, including the popularly used penicillin, cephalosporin, and their derivatives, are the most widely used antibiotics in the world.1,2 Besides their clinical applications, β-lactam antibiotics have been used in the stockbreeding industry to prevent and treat infectious diseases in cattle worldwide. However, the abuse of antibiotics has raised significant food safety issues such as allergic reactions and the imbalance of intestinal microflora caused by the residual antibiotics from the contaminated meat and milk products.3−5 More seriously, drug resistance can be transferred to human which brings about tough clinical issues.6 To ensure the safety and quality of milk products, governments have set maximum residue limits for β-lactams in the dairy industry (amoxicillin, 10 μg/kg; ampicillin, 10 μg/kg; benzylpenicillin, 4 μg/kg in China). However, sometimes dairy industries use β-lactamases (Blas) to degrade β-lactam in milk.7,8 Blas are a group of enzymes that can specifically hydrolyze the β-lactam ring in antibiotics such as penicillin and cephalosporin to deactivate their antibacterial properties. This illegal practice may bring new risk to the health of consumers because the safety of Bla has not been fully evaluated.9 Furthermore, the addition of Bla would make it possible to conceal and thus encourage the abuse of antibiotics during milk production, storage, and transport.10 More seriously, many types of Blas have been simultaneously added into milk, thus it is urgent to detect all possible Blas in milk to prevent this illegal practice. Conventional methods for detection of Bla include the cylinder plate method,11 highperformance liquid chromatography, and mass spectrome-

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try,12,13 electrochemical biosensor,14 polymerase chain reaction(PCR),15 fluorescent imaging,16 and the acidimetric and iodometric methods.17 Although many of these methods yield outstanding results, they are typically time- and laborconsuming, limiting their use in on-site and high-throughput screening. Recently, newly developed methods based on gold nanoparticles18 and oligonucleotide microarrays19 for detecting Bla in milk were reported. However, these methods require complex equipment and tedious procedures for handling data. In addition, these conventional methods cannot simultaneously detect different types of Blas in one sample. Thus, it is urgently needed to develop a simple and rapid method for point-of-care (POC) detections of all possible Blas in dairy industry. In previous studies, we developed a series of probes composed of fluorogenic groups and β-lactam, as substrates for specifically imaging β-lactamase gene expression20 and in vivo imaging of Bla activity.21,22 We also developed probes capable of reacting with specific Blas expressed on pathogens to image tuberculosis in vivo23 and identify antibiotics-resistant pathogens such as tubercle bacilli.24,25 To meet the challenges of rapid screening and detecting Blas in dairy industry, in this study we design Tokyo Green-tethered β-lactam (CDG-1), a nonfluorescent probe that can react with a broad spectrum of Blas to yield fluorescent products to indicate Received: March 22, 2016 Accepted: May 5, 2016

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DOI: 10.1021/acs.analchem.6b01122 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry the presence of Blas. In addition, we combine the immunomagnetic separation technique with this broad-spectrum probe to enhance the sensitivity and selectivity for the detection of Blas. To achieve point-of-care detection, we also combine this probe with a smartphone that allows convenient readout. We synthesized a cephalosporin-based fluorogenic substrate CDG-1 which contains Tokyo Green at the 3′-position that is initially nonfluorescent.24 Because the direct coupling of Tokyo Green at its phenolic position with cephalosporin at the 3′position is not stable enough,24 a benzyl ether linker was thus introduced between the 3′-position of the lactam and Tokyo Green to increase its stability. The cephalosporin structure in CDG-1 could be recognized and hydrolyzed by Bla. The opening of the β-lactam ring would trigger spontaneous fragmentation, leading to the cleavage of the ether bond at the 3′-position and subsequent release of free Tokyo Green, with a fluorescence emission at 525 nm when excited at 490 nm (Scheme 1). The recovery of the fluorescence of Tokyo Green

Figure 1. Universality and specificity of the CDG-1 probe to Blas in PBS solution (with 3% skim milk powder): (A) penicillinase, (B) Cpase, and (C) GM enzyme were used to evaluate its universality in detecting Blas. (D) HRP, (E) ALP, and (F) amylase were employed to investigate its specificity to Blas. All enzymes were added into blank milk samples.

Scheme 1. Principle of the Universal Fluorogenic Probe CDG-1 for the Fluorescent Detection of Blas

(Figure 1F) into CDG-1 solutions (1 μM). The fluorescence intensity of the CDG-1 probe kept consistently low even though the concentration of the enzymes rose gradually. These results indicate that CDG-1 not only has good response to different types of Blas but also has specificity to the Blas over other enzymes, owing to the unique cephalosporin structure of CDG-1. We optimized both the reaction time and the concentration of CDG-1. We optimized the reaction time of the CDG-1 probe with Blas by testing the reactivity of CDG-1 (1 μM) with TEM-1 (a typical type of penicillinases, 1 U/mL). The fluorescence intensity of CDG-1 increases as the reaction time increases (Figure S1A). After 15 min of reaction, the intensity of the fluorescence reached a plateau, being 10-fold of the initial fluorescence intensity (t-test, P < 0.001). We recorded the results at the time point of 15 min in the following experiments. We also optimized the concentration of CDG-1 by varying the concentrations of CDG-1 under different concentrations of TEM-1. Varying the concentration of the TEM-1 at 10−3, 10−2, 10−1, 1, or 10 U/mL, we added different concentrations of CDG-1 (0.1, 0.2, 0.4, and 0.8 μM) into the TEM-1 solutions separately. We measured the fluorescence intensities of the reaction mixture and found that the change of fluorescence intensity (ΔFI) increased with the increase of the concentration of CDG-1 from 0.1 μM to 0.4 μM (Figure S1B). However, when the concentration of the CDG-1 rose to 0.8 μM, the ΔFI was lower than that at 0.4 μM. Meanwhile, the fluorescence intensity of 0.1 μM CDG-1 is relatively low (156 a.u.). These results indicate that the optimum concentration of the CDG-1 for this reaction is 0.4 μM. After the optimization of CDG-1 concentration and reaction time, we used this fluorescent sensor to detect Blas in spiked milk samples to show that our sensor can both broaden the spectrum for detection and decrease the time required for the test. Different concentrations of TEM-1 and Cpase were added into the blank milk samples, respectively. The ΔFI value increased when the concentration of TEM-1 changed from 10 to 3 U/mL to 10 U/mL (Figure 2A), and the ΔFI rose significantly with the increase of the Cpase in the range of 5 × 10−3 to 10 U/mL (Figure 2B). We compare this antibody-free method with the commercial enzyme-linked immunosorbent assay (ELISA) kits to detect Blas in milk samples. The commercial ELISA kits (TEM-1-specific) can detect 10−3 U/

indicates the existence of the Bla. Because of the β-lactam ring in this probe, almost all the Blas could catalyze it to generate fluorescent products, making this probe capable of detecting broad-spectrum Blas. This unique property provides an effective strategy for detecion of all possible Blas in milk. Since the fluorescent intensity of the product relates to the amount of Blas, this method enables one-step detection of the total Blas in the milk. The CDG-1 probe is broad in-spectrum and shows good specificity to Blas. Penicillin and cephalosporin are widely used in the dairy industry, resulting in high-levels of these antibiotics in milk. Blas have been illegally added to break down these antibiotics to disguise the added antibiotics. We first chose a penicillinase (TEM-1), a cephalosporinase (Cpase), and golden magnolia (GM, a mixture of Blas, which is difficult to assay) enzymes to demonstrate the broad-spectrum of CDG-1. The fluorescence intensity of CDG-1 (1 μM) increases when the concentration of penicillinase (Figure 1A), Cpase (Figure 1B), and GM (Figure 1C) varied from 0 U/mL to 0.1 U/mL. This result shows that CDG-1 is a broad-spectrum probe for detecting various Blas. Meanwhile, we chose other encountered enzymes to test the specificity of the probe, including horseradish peroxidase (HRP), alkaline phosphatase (ALP), and amylase. We tested if other unrelated enzymes can react with CDG-1 by adding increasing concentration (0−0.1 U/ mL) of HRP (Figure 1D), ALP (Figure 1E), and amylase B

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Figure 2. Results of fluorescent sensor and ELISA for detection of TEM-1 and Cpase in spiked milk samples. The concentrations of TEM-1 (or Cpase) were 10−4, 5 × 10−4, 10−3, 5 × 10−3, 10−2, 5 × 10−2, 0.1, 0.5, 1, 5, and 10 U/mL. We define the threshold value (dashed blue lines in each diagram) as follows: the corresponding concentration that generates a positive signal 3 times greater than the standard deviation of the blank samples.

mL of TEM-1 (Figure 2C), but it cannot detect Cpase (Figure 2D). When different types of Blas are simultaneously added into the milk samples, our fluorescent sensor, CDG-1, can probe all the Blas in the samples. By contrast, one commercial ELISA kit can only detect one specific type of Blas. In addition, our method is finished in one step within 15 min. By contrast, ELISA takes at least 4 h and involves complex protocols (multiple steps of washing and so forth). Compared with ELISA, this fluorescence probe-based method is superior for rapid screening of Blas in milk samples because of its speediness (one-step detection within 15 min) and broadspectrum applicability. The CDG-1 probe is capable of detecting broad-spectrum Blas since all Blas may hydrolyze the β-lactam ring. In the realworld application, an issue is that how to distinguish different types of Blas added into milk. To detect one specific type of Blas and simultaneously improve the sensitivity, we combined this probe with immuno-magnetic separation (IMS) technique to detect Blas in the milk samples. The Bla-antibody-bearing magnetic beads (MBs) can specifically capture Bla from the milk sample to form MBs-antibody-Bla complex, which can be magnetically separated and resuspended in PBS (pH = 7.4, 0.01 M). Any presence of Bla will result in the enzymatic reaction to convert CDG-1 into Tokyo Green, yielding the product with green fluorescence (525 nm) at an excitation of 490 nm (Figure 3A). Taking TEM-1 as an example, we studied the sensitivity and linear range of the fluorescent immuno-magnetic separation (FIMS) sensor for detection of TEM-1. The ΔFI of the mixture increased when the concentration of TEM-1 increased from 0 U/mL to 102 U/mL, and the ΔFI rose greatly with the increase of the TEM-1 in the range of 5 × 10−3 to 10 U/mL. The enzymatic reaction saturated when the concentration of TEM-1 reached 102 U/mL (Figure 3B). We selected 5 × 10−3 to 10 U/mL range of TEM-1 to plot and fit the data. The linear regression analysis indicated that all the data are evenly distributed along the plotted line with a R2 value of 0.98 (Y = 1295.6X − 655.83, X = lg[CTEM‑1concentration]) (Figure 3C). Within the range of 5 × 10−3 to 10 U/mL of TEM-1, the

Figure 3. (A) Principle of the FIMS sensor for Bla detection. Different antibodies that can specifically recognize different types of Blas were conjugated to the magnetic bead to prepare different magneticantibody conjugates. CDG-1 probe combines with IMS can construct the FIMS to capture and detect Blas in the milk sample. (B) The sensitivity of FIMS sensor for detection TEM-1, the concentrations of TEM-1 were 0, 10−5, 5 × 10−5, 10−4, 5 × 10−4, 10−3, 5 × 10−3, 10−2, 5 × 10−2, 0.1, 0.5, 1, 5, 10, and 100 U/mL. We define the threshold value (dashed blue line) as follows: the corresponding concentration that generates a positive signal 3 times greater than the standard deviation of the blank samples. (C) The linear range of the FIMS sensor. (D) POC detection based on a UV lamp, and the visual result of the FIMS sensor for detection of TEM-1 whose concentrations were 0, 0.01, 0.02, 0.05, 0.1, and 0.2 U/mL. (E) Fluorescent signals collected by a smartphone were converted into the gray value using a color scan software APP and plotted against the TEM-1 concentration.

fluorescence intensity of the probe varied linearly which can serve as a standard curve for detecting TEM-1. The limit of detection (LOD) of TEM-1 in this FIMS is 10−5 U/mL (Figure S2). To achieve POC detection, we further simplified this assay by using a hand-held UV lamp as the source for excitation and a smartphone as the readout. An enhancement of the green fluorescence of the product is distinguishable by the naked eye when the concentration of TEM-1 is above 10−2 U/mL (Figure 3D). The fluorescent signal could be collected by a smartphone and further semiquantified by a color scan software (Figure 3E), and the semiquantitative result shows that a higher concentration of TEM-1 results in a brighter fluorescence. Thus, this method is suitable for POC detection of Blas in resource-limited regions where expensive apparatus are absent. We detected Bla in real milk samples to confirm the efficiency and reliability of the FIMS sensor. Two MBs were conjugated with two types of antibodies (anti-TEM-1 antibody and anti-Cpase antibody), respectively, which were added into the milk samples (1:1). If there were TEM-1 or Cpase in the samples, either or both of them could be captured by the MBs C

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without sample pretreatment. By using CDG-1 probe directly, we can detect Bla as low as 10−3 U/mL and 10−2 U/mL by fluorescent sensor and smartphone-based detection, respectively, within 15 min. The FIMS could further achieve a sensitivity of 10−5 U/mL and be finished within 30 min. The LOD of cylinder plate method and ELSIA for detection of Blas is 5 × 10−4 U/mL and 10−3 U/mL, respectively. The detection time of cylinder plate method and ELISA is both longer than that of FIMS. Thus, the CDG-1-based Bla detections can realize real POC testing. In conclusion, we report a novel CDG-1 probe which can specifically detect the broad-spectrum of Blas by enzymatic reaction and fluorescent readout. This method needs no complex sample pretreatment and multiwashing steps that allows for the POC detection of many types of Blas in milk samples. We believe that this fluorescent sensor has promising prospects not only in milk inspection but also in clinical and preventive medicine.

and eventually be detected. Three blank milk samples, 11 spiked milk samples, and 15 real milk samples were provided by the Chinese Academy of Inspection and Quarantine, in which the 15 real milk samples were detected to be positive by the cylinder plate method. First, we detected all the samples using a commercial ELISA kit (Wuxi JieShengJieKang Biotechnology Co., LTD). This ELISA kit is specific to the TEM-1. The blank milk samples present low levels of absorbance at a wavelength of 450 nm (Figure 4A). The OD450 nm value rose gradually with

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01122. Experimental details and supplementary figures, Figures S1−S3 (PDF)

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Figure 4. Detection of Blas in milk samples: (A) the ELISA results and (B) the detection results of the FIMS sensor. Samples 1st−3rd, the blank samples; samples 4th−18th, the real milk samples. All the samples were provided by the Chinese Academy of Inspection and Quarantine, and these samples were previously detected by the cylinder plate method. We define the threshold value as follow: 3 times the standard deviation from a number of measurements of the blank samples (3δblank), namely, the corresponding concentration that generates a positive signal 3 times greater than the standard deviation from the blank samples. Red lines indicate the threshold for the positive samples.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Fax: (+86)10-82545631. Phone: (+86)10-82545558. Author Contributions §

Y.C. and Y.X. equally contributed to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Ministry of Science and Technology of China (Grants 2011CB933201 and 2013AA032204), the National Science Foundation of China (Grants 21025520 and 21105018), and the Chinese Academy of Sciences (Grant XDA09030305) for financial support.

the increase of TEM-1 concentration from 10−2 to 102 U/mL in the spiked samples (Figure S3A). We found that only when the concentration of TEM-1 reached 10−2 U/mL or above, the samples could be recognized as positive using the ELISA kit. In contrast, the fluorescence signal started to increase when the concentration of spiked TEM-1 was 10−4 U/mL, which shows that the sensitivity of FIMS is better than that of ELISA. For the real milk sample, only 5 out of 15 samples (33%) could be detected as positive samples by the commercial ELISA kit (Figure 4A). In comparison, we detected the same samples using the FIMS sensor. For the real milk samples (sample 4 to sample 18), 93.3% samples (except sample 11) were detected as positive, demonstrating the high sensitivity of this approach (Figure 4B). The IMS strategy can enrich Blas in the enlarged volume of sample to enhance the sensitivity of this FIMS method. Compared with the commercial ELISA kit, the CDG1-based method has three advantages: (1) broad-spectrum reactivity, (2) high sensitivity, (3) POC detections. One commercial ELISA kit only works for one specific Bla, which dramatically limits its application in screening various Blas in complex samples since there may be more than one Blas in samples. In addition, ELISA typically needs 4−6 h while the approach developed in this study can be finished within 15 min

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