Fluorescence resonance energy transfer-mediated immunosensor

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Fluorescence resonance energy transfer-mediated immunosensor based on design and synthesis of the substrate of Amp cephalosporinase for biosensing Suimin Deng, Jing Wu, Kaina Zhang, Yike Li, Lina Yang, Dehua Hu, Yuhao Jin, Yun Hao, Xiangfeng Wang, Yuan Liu, Hailing Liu, Yiping Chen, and Meng-Xia Xie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02427 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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Analytical Chemistry

Fluorescence resonance energy transfer-mediated immunosensor based on design and synthesis of the substrate of Amp cephalosporinase for biosensing Suimin Deng,† Jing Wu,† Kaina Zhang,† Yike Li,† Lina Yang,† Dehua Hu,† Yuhao Jin,† Yun Hao,† Xiangfeng Wang,† Yuan Liu,† Hailing Liu,† Yiping Chen*‡ and Mengxia Xie*† † Analytical and Testing Center of Beijing Normal University, Beijing 100875, China. ‡ College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China. ABSTRACT: The traditional enzyme-linked immunosorbent assay (ELISA) has some disadvantages, such as insufficient sensitivity and low stability of the labeled enzyme, which limit its further applications. In this study, a more stable enzyme, Amp cephalosporinase (AmpC), was selected as the labeled enzyme, and its substrate was designed and synthesized. This substrate contained the cephalosporin ring core as the enzymatic recognition section and the structural motif of the 3-hydroxyflavone (3-HF) as the reporter molecule. AmpC can specifically catalyze the substrate and release 3-HF, which can enter the cavity of β-cyclodextrin (β-CD) on the surface of ZnS Quantum dots and form a fluorescence resonance energy transfer (FRET) signal amplification system. An AmpC-catalyzed, FRET-mediated ultrasensitive immunosensor (ACF immunosensor) for procalcitonin (PCT) was developed by combining the signal amplification system of the polystyrene microspheres and effective immune-based magnetic separation. The ACF immunosensor has high sensitivity and specificity for the detection of PCT; its linear range is from 0.1 ng mL-1 to 70 ng mL-1, and the limit of detection can reach 0.03 ng mL-1. The spiking recoveries of PCT in human serum samples range from 98.3% to 107%, with relative standard deviations ranging from 2.14% to 12.0%. This approach was applied to detect PCT in real patient serum samples, and the results are consistent with those obtained with a commercial ELISA kit.

Highly sensitive and stable assays for measuring low concentrations of biomarkers in clinical samples have become increasingly important in analytical science.1 The enzyme linked immunosorbent assay (ELISA)2, is a widely used traditional immunoassay in clinical diagnosis3, food safety4 and environmental monitoring5 because of its high throughput and relatively low cost.6 However, some disadvantages hinder further applications of this assay. ELISA requires labeled enzymes, usually horseradish peroxidase (HRP)7 or alkaline phosphatase (ALP)8, which are not stable at room temperature and require cryopreservation, limiting their application in hostile environments9. Furthermore, the sensitivity of the ELISA method sometimes cannot satisfy the need for the detection of trace targets in complex matrices.10 Therefore, it is an important challenge to search for a highly stable enzyme to improve the stability of the assay and construct an effective signal amplification system that matches the stable bioenzyme to enhance the sensitivity of conventional ELISA. Numerous studies have attempted to overcome these problems. Various novel kinds of enzymes have been adopted to improve the stability of ELISA, such as artificial nanoenzymes.11, 12 However, insufficient selectivity, low repeatability and unsatisfactory salt tolerability are common challenges facing most artificial nanozymes.13 Thus, it is also necessary to find other stable bioenzymes with high catalytic efficiency. Many strategies have been adopted to enhance the signal readout to improve the sensitivity of the assay.3, 14

Nanoparticles and bioconjugation strategies have been described as efficient signal readout and amplification systems, which provide an attractive method to improve the sensitivity of ELISA.14 Long and his coworkers conjugated tyrosinase with detection antibody and prepared dopamine (DA)-functionalized CdSe/ZnS quantum dots to determine α-fetoprotein (AFP) levels.15 Our group has developed an immunosensor with conjugating anti-S. enterica antibody and HRP simultaneously on the gold nanoparticles, to recognize S. enterica targets and amplify signal readout.16 β-lactamase is a natural bioenzyme that widely exists in tubercle bacilli, and it can greatly degrade lactam antibiotics, which is the main reason that tubercle bacilli have drug resistance.17 Due to its high stability at room temperature and efficient degradation of antibiotics,18 β-lactamase has been widely used as an antibiotic remover to degrade antibiotic residues in the environment.19 Furthermore, the β-lactamase strain with high production can be selected in many gramnegative bacteria by natural screening, UV mutation and resistant screening, which make this enzyme less expensive than other natural enzymes.20

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Scheme 1 Schematic Illustration of ACF immunosensor for detection of PCT.

(A) Specific binding between IMBs-Ab1-PCT and PS-Ab2/(AmpC)n, and formation of IMBs-Ab1-PCT-Ab2-PS-(AmpC)n complex. (B) Enzymatic reaction process of MX-66 by AmpC enzyme on the surface of the complex, and formation of FRET signal amplification system between the reporter molecule (3-HF) and β-CD-ZnS QDs. The main action site of β-lactamase is the β-lactam structure motif in lactam antibiotics,21 which means that its specific substrate can be designed according to our requirements. Xie and his coworker prepared a fluorogenic probe with a structure similar to that of carbapenem to determine carbapenemase activity and carbapenemase-producing organisms.22 Our group has also developed a point-of-care assay for the detection of β-lactamase in milk with a universal fluorogenic probe (Tokyo Green-tethered β-lactam).23 Therefore, with the design and synthesis of a suitable substrate, β-lactamase may be a stable labeled bioenzyme in immunoassays. An effective signal amplification system is an important path for enhancing the sensitivity of immunoassays. Fluorescence resonance energy transfer (FRET) has been widely used in the analytical science and biological imaging24 as a powerful signal amplification strategy. Compared with other signal amplification assays, FRET has high fluorescence transfer efficiency to enhance the signal and excellent designability that can satisfy different requirements for biosensors.25, 26 Our group has developed fluorescent FRET probes for the determination of glucose and megestrol acetate based on β-cyclodextrinmodified ZnS quantum dots (β-CD-ZnS QDs) and natural pigment 3-hydroxyflavone (3-HF) and neutral red (NR),27, 28 respectively. The results showed that ZnS-QDs can strongly improve the fluorescence emission intensities of the reporter molecule through the FRET process. Thus, we hope that FRET can also be used as an effective signal amplification tool to improve the sensitivity of conventional ELISA. To address the challenges of stability and sensitivity facing conventional ELISA, a novel immunoassay based on βlactamase as a labeled enzyme and FRET signal amplification system was investigated and tested for the detection of procalcitonin (PCT) (see Scheme 1). For this purpose, Amp cephalosporinase (AmpC, belonging to Ambler class C βlactamase)29 was selected as a labeled enzyme, and an AmpCspecific substrate composed of 3-etherflavone-tethered βlactam was designed and synthesized. To improve the

sensitivity of the assay, AmpC and the detection antibody for PCT were covalently attached to polystyrene (PS) microspheres simultaneously. Because there are more carboxyl binding sites on the surface of PS microspheres,30 more AmpC can be bound on the surface of PS and compose the first enzymatic signal amplification system. AmpC can catalyze the substrate to release the reporter molecule, 3-hydroxyflavone (3-HF), which can form a FRET system with β-CD-ZnS QDs.27 In this FRET system, 3-HF was used as the energy acceptor, and β-CD-ZnS QDs acted as the energy donor, which can significantly enhance the fluorescence intensity of the 3-HF (see Scheme 1B). Thus, the fluorescence intensity of 3-HF could be used as the signal readout. Combined with the immunomagnetic separation (IMS) technique,31 an AmpC-catalyzed, FRET-mediated cascade reaction immunosensor (ACF immunosensor) was developed. The immunosensor was successfully applied to detect PCT in real samples and exhibited acceptable accuracy.

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EXPERIMENTAL SECTION

Synthesis of MX-66 substrate. To a mixture solution of compound 1 (4-methoxybenzyl (6R, 7R)-7-amino-3(chloromethyl)-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2carboxylate hydrochloride, ACLE HCl, 4.05 g, 10 mmol) and pyridine (4.8 g, 60 mmoL) in DCM (dichloromethane, 150 mL), phenylacetyl chloride (3.1 g, 20 mmoL) was added, and the reaction mixture was stirred at room temperature (RT) for 2 hours. Then, the reaction mixture was washed with saturated NaHCO3 (150 mL x 3) three times, and brine (150 mL), dried over anhydrous Na2SO4, concentrated and purified by flash column (C18 reversed phase, MeCN/H2O as eluent) to give product 2 (4.5 g, yield 92%) as a white solid. Then product 2 (4.5 g, 9.2 mmoL) was mixed with 3hydroxyflavone (2.3 g, 9.6 mmoL), K2CO3 (1.65 g, 11.9 mmoL) and KI (150 mg, 0.9 mmoL) in acetone (150 mL), stirring at 40 oC for 24 h. Condensed the mixture and the residues were dissolved in ethyl acetate (20 mL). The mixture was washed


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with brine (200 mL), dried over anhydrous Na2SO4, concentrated and purified by flash column (C18 reversed phase, MeCN/H2O as eluent) to give product 3 (3.0 g, yield 47%) as a yellow solid. Then, the product 3 (3.0 g, 4.3 mmoL) was dissolved in DCM (90 mL) and cooled to 0 oC, and TFA (trifluoroacetic acid, 9 mL) was added dropwise, and the resulting mixture was stirred at RT for 0.5 h. The mixture was concentrated at RT under reduced pressure and the residue was triturated with CH3CN/DCM (v/v = 1/3, 40 mL) and filtered to afford compound 4 (MX-66, 500 mg, yield 20%) as a yellow solid. 1H NMR (500 MHz, DMSO-d ) δ 13.66 (s, 1H), 9.01 (d, 6 J=8.5Hz, 1H), 8.04 (d, J=8.0Hz, 1H), 7.97 (s, 2H), 7.76 (t, J=7.0Hz, 1H), 7.70-7.50 (m, 1H), 7.52 (s, 3H), 7.44 (t, J=7.5Hz, 1H), 7.24-7.15 (m, 5H), 5.52-5.48 (m, 1H), 4.93-4.70 (m, 2H), 4.74 (d, J=4.8Hz, 1H), 3.53-3.40 (m, 4H). 13C NMR (126 MHz, DMSO-d ) δ 174.39, 171.46, 164.77, 6 163.64, 156.39, 155.30, 139.74, 136.32, 134.63, 131.41, 130.82, 129.49, 129.17, 129.11, 128.68, 126.95, 125.59, 123.96, 118.93, 71.25, 59.37, 57.94, 42.03, 40.48, 40.31, 40.15, 39.98, 39.81, 39.65, 39.48, 26.31. HRMS (ESI+) m/z calcd for C31H24N2O7S [M]+ 568.13042, found 569.13802. MX-66: (6R,7R)-8-oxo-3-(((4-oxo-2-phenyl-4H-chromen3-yl)oxy)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid. We commissioned 9dingchem Co., Ltd to synthesize the MX-66. Characterization of the ACF immunosensor. The detection of PCT was carried out as follows: The Ab1conjugated immuno-magnetic balls (IMBs-Ab1) complex and the Ab2 and AmpC-conjugated polystyrene microspheres (PSAb2/(AmpC)n) complex (nAmpC/nAb2=100:1) were first diluted to 0.25 mg mL-1 and 1.0 mg mL-1 with PBS buffer, respectively. Then, 50 μL of 0.25 mg mL-1 IMBs-Ab1 complex were added into 450 μL of PCT solution with various concentrations (0.0960, 0.288, 0.864, 2.59, 7.78, 23.3 and 70.0 ng mL-1, respectively) and reacted on a roller for 20 min at 37°C. Then, the IMBs-Ab1-PCT complex was separated by a magnetic separator and washed twice with 500 μL PBST buffer and resuspended in 500 μL PBS buffer. Fifty microliters of 1.0 mg mL-1 PS-Ab2/(AmpC)n complex was subsequently introduced and reacted on a roller for 20 min at 37°C. The unreacted complex was removed by a magnetic separator, and the double sandwich complex was washed with 500 μL PBST buffer three times and transferred to a new tube in which 400 μL of MX-66 solution (1.0*10-4 mol L-1) was added, and the mixture solution was incubated for 20 min on a roller at 37°C. Then, the double sandwich complex was removed by a magnetic separator, and 25 μL of β-CD-ZnS QDs (0.711 mmol L-1) was introduced into the solution. The fluorescence intensities at 530 nm were collected at an excitation wavelength of 350 nm. The final concentrations of MX-66 and β-CD-ZnS QDs were 75.5 μmol L-1 and 44.4 μmol L-1, respectively. With similar procedures, the concentration of PCT was fixed to 1.40 ng mL-1, and the influence of the PS-Ab2/(AmpC)n complex with various molar ratios of AmpC to Ab2 on the surface of PS microspheres (nAmpC/nAb2=30:1, 70:1, 100:1, 130:1, 160:1, respectively) on the fluorescence intensities was investigated.

To determine the influence of the enzymatic reaction time on the results, enzymatic reaction times of 5, 10, 20 and 30 min were tested, and other procedures were as described above. For the specificity of the immunoassay, seven control experiments were performed. Three common interference components (CRP, IL-6 and human IgG) and a mixture of PCT and blank samples were selected to investigate the selectivity and specificity of the ACF immunosensor. The concentrations of CRP, IL-6 and human IgG were 100 ng mL-1 in PBS buffer, and the concentration of PCT was 7.78 ng mL-1. Employing the same experimental procedures described above, the seven samples were assayed, and each sample was repeated three times. Spiking recovery of PCT in human serum sample. To remove the adipose tissue, the healthy human serum was centrifuged at 10000 rpm for 20 min three times. Then, 50 μL of PCT at various concentrations (0.432, 1.30, 3.89, 11.7, 35.0 and 70.0 ng mL-1) was spiked in 350 μL of the above centrifuged serum. The spiked samples were assayed with the procedures described above. The final concentrations of PCT were 0.273, 0.547, 1.09, 2.19, 4.38 and 8.75 ng mL-1. Real patient samples detection. The PCT levels in 14 real patient serum samples (obtained from Beijing Friendship Hospital with patient consent) were detected with the ACF immunosensor following the procedures described above, and each sample was assayed three times. For verification, a commercial ELISA kit was also utilized to detect the PCT levels of the same 14 samples with the recommended method of the kit. Briefly, 50 μL of standard sample or real sample was added to the 96-well plates along with 100 μL of HRP-labeled PCT detection antibody. The 96-well plates were incubated at 37°C for 1 h and washed five times with washing buffer, substrates A and B were added to the plates, and the plates were incubated at 37°C for 15 min. Finally, 50 μL of the stopping solution was added to the plates, and the UV absorbance of the samples was detected at 450 nm. Reagents and instruments, and other experiments procedures were described in supporting information.

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RESULTS AND DISCCUSSION

Design and synthesis of the substrate for the AmpC (MX66). To develop an enzymatic reaction system, the substrate for the AmpC enzyme was designed as MX-66 (C31H24N2O7S), which contained the β-lactam ring and cephalosporin ring as the enzymatic recognition section and the structural motif of a natural green dye, 3-HF, as the reporter molecule. The synthetic route of MX-66 is shown in Fig. 1. The structure of the product was confirmed by highresolution mass spectrometry (HRMS, Fig. S2), Fourier transformed infrared spectroscopy (FTIR, Fig. S1 and Table S1) and nuclear magnetic resonance (NMR, Fig. S3-S5) approaches. In the HRMS spectra of the product, there are two main peaks at 569.13802 Da (m/z) and 591.12004 Da (m/z), which represent the M+H+ and M+Na+, respectively. From the exact mass and isotopic profiles in the HRMS spectra, the molecular formula of the product was deduced as C31H24N2O7S, which was the same as that of the designed target MX-66 (the theoretical exact mass of the target MX-66 is 568.13042 Da.). The characteristic FTIR absorption bands of the main functional groups are described in Table S1, and these findings suggested



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O

H 2N

HCl S

H N

O

N

Pyridine

Cl

O O

HO

Cl

O

1

O

O

S

Acetone

O O

O

N

O

H N

O O

O

K2CO3, KI

Cl

2

O H N

N O

DCM

O

Ph

S

O

Ph

TFA

O

DCM

O

S

O

N

O

O

OH Ph

O O

O

4 (MX-66)

3 Figure 1. The synthetic route of the substrate MX-66. the presence of the carbonyl group in the β-lactam ring (a fourmembered ring, νc=o, 1774 cm-1), the other carbonyl groups in the amide and carboxyl groups and the 3-HF motif (1630-1661 cm-1), and the monosubstituted benzene ring (759 cm-1, 695 cm1). Various NMR methods, including 1H NMR, 13C NMR and heteronuclear single quantum coherence (HSQC), were applied to confirm the structure of the product, and the detailed data are shown in the experimental section and supporting information. Catalytic reaction of MX-66 by AmpC and formation of the FRET system. The mechanism of the catalytic reaction has been described in Scheme 1. When AmpC enzyme exists, the active site (serine) of the enzyme will specifically attack the amide bond in β-lactam ring, and lead to the breakage of the amide bond.21 Then, the opening of the β-lactam ring will result the electron transfer and the structural arrangement of the hydrogenated thiazine hexagonal ring, which lead to the release of the 3-hydroxyflavone (3-HF). To form the FRET system, the β-CD ZnS QDs were synthesized according to the procedures in our previous report,28 and their properties were characterized (Fig. S6-S7). Fig. 2 shows the fluorescence spectra of MX-66

Figure 2. Fluorescent characterization of the enzymatic reaction and FRET system. (A) The fluorescence spectra of MX-66 (a), β-CD-ZnS QDs (b), β-CD-ZnS QDs + AmpC (c), MX-66 + β-CD-ZnS QDs (d), MX-66 + AmpC (e) and MX-66 + AmpC + β-CD-ZnS QDs (f), excited at 350 nm. (B) The fluorescence intensities at 530 nm of MX-66 (a), β-CD-ZnS QDs (b), β-CD-ZnS QDs + AmpC (c), MX-66 + β-CD-ZnS QDs (d), MX-66 + AmpC (e) and MX-66 + AmpC + β-CD-ZnS QDs (f), excited at 350nm. The final concentrations of MX-66, AmpC and β-CD-ZnS were 5.83 μmol L-1, 3.26 μmol L-1 and 17.8 μmol L-1, respectively.

(a), β-CD-ZnS QDs (b), β-CD-ZnS QDs + AmpC (c), MX-66 + β-CD-ZnS QDs (d), MX-66 + AmpC (e) and MX-66 + AmpC + β-CD-ZnS QDs mixture(f) upon excitation at λ=350 nm. The substrate of the AmpC enzyme, MX-66, has almost no fluorescence emission, and the fluorescence emission band of the β-CD-ZnS QDs was at approximately 430 nm. AmpC enzyme has little effect to fluorescence emission of the β-CDZnS QDs. When MX-66 was mixed with the AmpC enzyme, the 3-HF was released from the MX-66 under catalysis of AmpC, which has a weak fluorescence emission band at approximately 510 nm. With the addition of the β-CD-ZnS QDs, the fluorescence intensity of the system increased significantly, accompanying with the red shift of the emission band to 530 nm, which was similar to the results obtained after mixing the 3-HF with β-CD-ZnS QDs (Fig. S7B). The results demonstrated that the 3-HF was successfully released from the substrate MX-66 with the addition of the AmpC enzyme, and the FRET system between the 3-HF and β-CD-ZnS QDs was formed. To confirm the mechanism of the catalytic reaction by AmpC enzyme, the HRMS spectra of the substrate before and after the enzymatic reaction were collected. As shown in Fig. 3B, most of the substrate reacted with the addition of the enzyme, the peak of M+H+ nearly disappeared, and the peak of M+Na+ significantly decreased. Meanwhile, several new peaks (m/z: 239.07028 and 261.05244) emerged. From the accurately measured molecular mass and isotopic pattern, the new component with m/z 239.07028 can be proposed to be 3-HF (M+H+, the molecular formula of 3-HF is C15H10O3, m/z: 238.06299), while the peak with m/z 261.05244 was 3-HF (M+Na+). With increasing concentrations of the AmpC enzyme, the peak of M+Na+ (m/z: 591.12004) also disappeared, which indicated that the enzymatic reaction of the substrate was complete (see Fig. 3C). Having fixed the concentration of the substrate for the AmpC enzyme, the influence of the concentrations for the β-CD-ZnS QDs on the signal output of the enzymatic reaction system was investigated (see Fig. S8A-B). The fluorescence intensities (at 530 nm) of the reporter molecule (3-HF) gradually increased with increasing concentrations of the β-CD-ZnS QDs, and the fluorescence signal tended to reach its maximum when the


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Figure 3. Characterization of the enzymatic reaction for MX66 by HRMS. The HRMS spectra of (A) MX-66, (B) MX-66 catalyzed by 0.05 mg ml-1 MBs-AmpC complex and (C) MX66 catalyzed by 0.50 mg ml-1 MBs-AmpC complex. The final concentration of MX-66 solution was 9.0 μmol L-1.

concentration of the β-CD-ZnS QDs was higher than 44.5 μmol L-1, which signified that most of the 3-HF had entered the cavity of the β-CD on the surface of the ZnS-QDs. To determine the suitability of the catalytic reaction system to serve as an immunosensor with 3-HF as a reporter molecule, the effect of the AmpC concentrations on the release of 3-HF from the substrate was investigated. The fluorescence intensities of the catalytic reaction system (with substrate of 5.33 μmol L-1) with various concentrations of the AmpC enzyme, ranging from 0.0283 μmol L-1 to 14.5 μmol L-1, with and without the β-CD-ZnS QDs were studied (see Fig. S8C). It can be seen from the curves that the fluorescence intensities initially increased significantly with increasing concentrations of AmpC with β-CD-ZnS QDs and then tended to reach constant values gradually when the concentration of the enzyme further increased, which indicated that the catalytic reaction had completed. Without the β-CD-ZnS QDs, the curves presented a similar tendency, while the fluorescence intensities were notably weak. Furthermore, the relationship between the fluorescence intensities of the system and the logarithm of the AmpC concentrations indicated that a satisfactory linear relationship can be obtained when the concentration of the AmpC enzyme ranged from 0.226 μmol L-1 to 3.62 μmol L-1 (see Fig. S8D). The results also implied that the substrate of the AmpC enzyme has the potential to develop an enzymatic reaction immunosensor and that the FRET system between the reporter molecule, 3-HF, and the β-CD-ZnS QDs can significantly enhance the signal output. The stability of the AmpC enzyme was examined and compared with that of HRP. The UV absorbance band of the

nitrocefin can be moved from 390 nm (yellow) to 492 nm (pink) after hydrolyzing nitrocefin by the AmpC enzyme,32 which can be utilized to characterize the activity of the AmpC enzyme. HRP can catalyze 3,3',5,5'-tetramethylbenzidine (TMB), and the enzymatic activity of HRP can be estimated by the UV absorption intensity of TMB at 450 nm.33 The enzymatic activities of the AmpC and HRP enzymes with the storage time of the enzymes are investigated (see Fig. S9). The results showed that the activity of the AmpC enzyme remained stable after storage at room temperature for 5 days and only slightly decreased for 11 days. However, the activity of the HRP enzyme was significantly reduced after storage for 2 days under the same conditions. This result indicated that the stability of the AmpC enzyme is clearly higher than that of HRP, which can greatly improve the stability of the immunoassay when AmpC is used as the labeled enzyme. ACF immunoassay for PCT detection. PCT is the precursor of calcitonin, consisting of 116 amino acids.34 PCT is an important bacterial inflammatory biomarker that can be elevated after suffering severe bacterial, fungal, parasitic infections, sepsis and multiple organ failure.35 Generally, antibiotic therapy will be adopted when the concentration of PCT exceeds 0.50 ng mL-1.36 Hence, the ultrasensitive and accurate detection of PCT in serum is significant for early diagnosis and prognosis therapy. To develop an ultrasensitive immunosensor for determination of PCT based on the above enzymatic reaction system, a multiplex signal amplification strategy has been applied. We coupled the AmpC enzyme and PCT detection antibody to the surface of PS microspheres simultaneously to form a PS-Ab2/(AmpC)n complex as another signal amplification system (as shown in Scheme 1). A large number of carboxyl binding sites on the surface of PS microspheres led to high efficiency for the conjugation of enzyme and antibody proteins.37 The effect of signal amplification can be adjusted by the mole ratios of the enzyme to the antibody proteins. For convenient sample pretreatment, the PCT capture antibody has been bound to the surface of magnetic nanoparticles to form the immunomagnetic beads (IMBs-Ab1), which can specifically recognize and enrich the PCT in sample solution to form the IMBs-Ab1-PCT complex. The IMBs-Ab1-PCT complex can combine with the PCT detection antibody on the PS-Ab2/(AmpC)n complex, forming a double antibody sandwich system (IMBs-Ab1-PCT-Ab2PS/(AmpC)n). The AmpC enzyme on the surface of PS microspheres can catalyze the substrate MX-66 and release the reporter molecule 3-HF, which was related to the quantities of the PCT in the sample. Therefore, an AmpC-catalyzed, FRETmediated immunosensor was developed for the measurement of PCT in serum samples combined with the immunomagnetic technique. The PS-Ab2/(AmpC)n complex, the IMBs-Ab1 complex and their conjugate were characterized by scanning electron microscopy (SEM) (see Fig. S10). The profiles of the IMBs-Ab1 were changed compared with that of magnetic nanoparticles before conjugation with the PCT capture antibody (Ab1) (see Fig. S10A-B). After coupling the AmpC enzyme and detection antibody, the profiles of the PS-Ab2 /(AmpC)n particles became notably rough (see Fig. S10C-D). The size distribution of nanoparticles was characterized by dynamic light scattering (DLS), and the results showed that the average



Figure 4. The performances of the ACF immunosensor. (A) The fluorescence emission spectra of the ACF immunosensor with various concentrations of PCT. (B) The linear regression curve between the fluorescence intensities at 530 nm of the ACF immunosensor and logarithm of PCT concentrations. (C) The selective of ACF immunosensor for detection of PCT. The final concentrations of PCT, IL-6, CRP and Human IgG were 7.78 ng ml-1, 100 ng ml-1, 100 ng ml-1 and 100 ng ml-1, respectively. (D) The PCT levels in 14 real patient samples determined by the ACF immunosensor and conventional ELISA kit (n=3).

particle diameters for MBs, IMBs-Ab1, PS microspheres and PS-Ab2/(AmpC)n complexes were 1067.4 nm, 1222.4 nm, 1039.2 nm and 1298.6 nm, respectively (see Fig. S11). The diameters of the IMBs-Ab1 and PS-Ab2/(AmpC)n complexes notably increased compared with those of MBs and PS microspheres, which suggested that the AmpC enzyme and the antibody were successfully coupled to the surface of relative beads. From the subtraction spectra between the diffuse reflection FTIR spectra of the PS-Ab2/(AmpC)n and that of PS microspheres (see Fig. S12), there are two main absorption bands at about 1652 cm-1 and 1548 cm-1, which can be ascribed to the amide I band and amide II band of proteins, respectively. The FTIR results further confirmed that the proteins were coupled to PS microspheres. The SEM image of the double antibody sandwich system formatting between the IMBs-Ab1PCT and the PS-Ab2/(AmpC)n, clearly demonstrated the formation of the IMBs-Ab1-PCT-Ab2-PS-(AmpC)n complex (see Fig. S10E). The molar ratio of the AmpC enzyme to the PCT detection antibody (Ab2) on the surface of PS microspheres would significantly influence the efficiency of signal amplification. Therefore, the molar ratios of AmpC to Ab2 on the surface of PS microspheres were optimized. Fixing the quantities of PCTAb2 (1.33 nmoL), and with the molar ratios of AmpC to Ab2 ranging from 30:1 to 160:1, a series of PS-Ab2/(AmpC)n complexes were prepared and applied to the ACF immunosensor for detection of 1.40 ng mL-1 PCT. The

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fluorescence intensities of the ACF immunosensor were clearly enhanced with increasing molar ratios of AmpC enzyme to Ab2 from 30:1 to 100:1 (see Fig. S13). The fluorescence intensities increased slightly, and the deviations increased when the molar ratios increased. Thus, we selected the optimal molar ratio of AmpC to Ab2 to be 100:1. The enzymatic reaction time of MX-66 by the AmpC enzyme was also optimized (see Fig. S14). The fluorescence intensities of the ACF immunosensor were relatively weak when the catalytic time was 5 min, and satisfactory fluorescence intensities of the system could be reached after catalyzing 20 min. Although the signal became slightly stronger when the reaction time was 30 min, it would be overly time-consuming and unsuitable for POC testing. Thus, we eventually selected 20 min as the best reaction time between AmpC enzyme and MX66 and applied it in subsequent experiments. Fig. 4A shows the fluorescence spectra of the ACF immunosensor, with the concentrations of PCT ranging from 0 to 70.0 ng mL-1. As shown in Fig. 4A, the fluorescence intensities of the band at 530 nm gradually enhanced with increasing concentrations of PCT in the buffer solution. The relationship between the response signal (fluorescence intensities at 530 nm) and the logarithm of PCT concentrations was plotted (see Fig. 4B), and the linear regression equation was y=1.92*105x+6.04*105 with regression coefficient R2=0.9919 in the concentration range from 0.1 to 70 ng ml-1. The limit of detection (LOD) was estimated as 0.03 ng ml-1 (LOD=3σ/S, where σ was the value of the standard deviation of the blank sample response, and S was the slope of the standard curve within the linear range of the low concentration, σ=154.14, S=15586, n=10). The selectivity of the ACF immunosensor has been estimated by introducing several common interference components. Creactive protein (CRP) is one of the inflammatory markers, as well as the most powerful predictor and risk factor for cardiovascular disease.38 Interleukin-6 (IL-6) is involved in the occurrence and development of many diseases, and its blood level is closely related to inflammation, viral infection and autoimmune diseases. Most importantly, IL-6 can indicate whether it is a bacterial infection.39 Human immunoglobulin G (human IgG) is the main antibody composition in human serum and plays a protective role in body immunity.40 Fig. 4C shows the fluorescence intensities at 530 nm of the ACF immunosensor for PCT (7.78 ng mL-1), CRP (100 ng mL-1), IL6 (100 ng mL-1), human IgG (100 ng mL-1), and the mixture of PCT with each of the three interference components. The fluorescence intensity for PCT was similar to those for the mixtures of PCT and each interference component, and the fluorescent signals of the three interference components were notably weak and similar to that of the blank sample. These results indicated that the interference components have minor effects on the detection of PCT and that the ACF immunosensor has high specificity. To evaluate the accuracy of the ACF immunosensor for the detection of PCT in human serum, various levels of PCT were spiked in healthy human serum to estimate their spiking recoveries and reproducibility. Considering that the clinical diagnostic standard to distinguish bacterial and nonbacterial inflammation is 0.5 ng mL-1, the spiking levels of PCT have been selected from 0.273 ng mL-1 to 8.75 ng mL-1. As shown in


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Table 1, the spiking recoveries of PCT in human serum ranged from 98.3% to 107% with relative standard deviations (RSD) from 2.14% to 12.0%, which satisfied the requirement for measuring the trace analytes in complex matrices. The results demonstrated that the ACF immunosensor could be applied for clinical analysis of biomarkers in human serum. The ACF immunosensor was applied to measure the levels of PCT in 14 human serum samples obtained from Beijing Friendship Hospital, and the results were confirmed with a conventional commercial ELISA kit. Fig. 4D and Table S2 show the PCT levels of the samples measured by the two approaches. The results indicated that the PCT levels of the samples (No. 1, 3, 8, 10, 12, and 14) obtained by the ACF immunosensor have excellent agreement with those obtained by the conventional ELISA kit (see Fig. S15 for correlation analysis and paired t-test), which further illustrated that the developed immunosensor has high feasibility to quantify the biomarker in human serum. The PCT levels of other serum samples (No. 2, 4, 6, 9, 11, and 13) were beyond the linear range of the conventional ELISA kit (from 0.5 to 16 ng mL-1); therefore, the accurate PCT levels of these samples cannot be obtained by the ELISA kit. However, the ACF immunosensor has higher sensitivity and a wider linear range and can provide precise and accurate PCT levels of these samples (see Table S2).

sensitive analysis of low-abundance biomarkers in real clinical samples.

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Fifteen supplementary figures and three table showing characterization and experimental data (PDF).

■ AUTHOR INFORMATION Corresponding Author *† E-mail: [email protected] *‡ E-mail: [email protected]

Notes The authors declare no competing financial interest

■ ACKNOWLEDGMENT We thank the National Natural Science Foundation of China (81671784) and the Beijing Nova Program (Z181100006218017) for financial support.

■ REFERENCES Table 1. Results of detection of spiked PCT in healthy human serum by ACF immunosensor (n=3) Spiked value (ng ml-1)

Measured value (ng ml-1)

Recovery (%)

RSD (%)

0.273

0.293 ± 0.021

107

7.33

0.547

0.573 ± 0.061

105

10.7

1.09

1.16 ± 0.12

106

10.3

2.19

2.19 ± 0.15

100

6.89

4.38

4.30 ± 0.09

98.3

2.14

8.75

8.76 ± 0.11

100

12.0

Compared with other platforms for detecting PCT, our method exhibits a wider linear range and comparable sensitivity and stronger selectivity (Table S3). The analysis time was shorter than that of conventional ELISA. This ACF immunosensor shows strong potential for the clinical detection of biomarkers.

■

CONCLUSIONS

In this study, an AmpC-specific fluorescent substrate was designed and successfully synthesized, and an AmpCcatalyzed, FRET-mediated immunosensor for measuring the biomarker PCT was developed. The results suggested that AmpC is more stable than the traditional immune-labelling enzyme (HRP) and that the AmpC-catalyzed FRET signal amplification system can effectively enhance the sensitivity of the immunosensor. The developed immunoassay has excellent specificity for PCT and can measure the PCT levels in serum samples with high sensitivity and accuracy. This work provides new findings for developing a stable immunosensor for highly

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For TOC only


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Scheme 1 Schematic Illustration of ACF immunosensor for detection of PCT. (A) Specific binding between IMBs-Ab1-PCT and PS-Ab2/(AmpC)n, and formation of IMBs-Ab1-PCT-Ab2-PS(AmpC)n complex. (B) Enzymatic reaction process of MX-66 by AmpC enzyme on the surface of the complex, and formation of FRET signal amplification system between the reporter molecule (3-HF) and βCD-ZnS QDs. 172x69mm (300 x 300 DPI)



Figure 1. The synthetic route of the substrate MX-66. 167x78mm (600 x 600 DPI)


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Figure 2. Fluorescent characterization of the enzymatic reaction and FRET system. (A) The fluorescence spectra of MX-66 (a), β-CD-ZnS QDs (b), β-CD-ZnS QDs + AmpC (c), MX-66 + β-CD-ZnS QDs (d), MX-66 + AmpC (e) and MX-66 + AmpC + β-CD-ZnS QDs (f), excited at 350 nm. (B) The fluorescence intensities at 530 nm of MX-66 (a), β-CD-ZnS QDs (b), β-CD-ZnS QDs + AmpC (c), MX-66 + β-CD-ZnS QDs (d), MX-66 + AmpC (e) and MX-66 + AmpC + β-CD-ZnS QDs (f), excited at 350nm. The final concen-trations of MX-66, AmpC and β-CD-ZnS were 5.83 μmol L-1, 3.26 μmol L-1 and 17.8 μmol L-1, respectively. 80x36mm (600 x 600 DPI)



Figure 3. Characterization of the enzymatic reaction for MX-66 by HRMS. The HRMS spectra of (A) MX-66, (B) MX-66 catalyzed by 0.05 mg ml-1 MBs-AmpC complex and (C) MX-66 catalyzed by 0.50 mg ml-1 MBsAmpC complex. The final concentration of MX-66 solution was 9.0 μmol L-1. 80x103mm (300 x 300 DPI)


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Figure 4. The performances of the ACF immunosensor. (A) The fluorescence emission spectra of the ACF immunosensor with various concentrations of PCT. (B) The linear regression curve between the fluorescence intensities at 530 nm of the ACF immunosensor and logarithm of PCT concentrations. (C) The selective of ACF immunosen-sor for detection of PCT. The final concentrations of PCT, IL-6, CRP and Human IgG were 7.78 ng ml-1, 100 ng ml-1, 100 ng ml-1 and 100 ng ml-1, respectively. (D) The PCT levels in 14 real patient samples determined by the ACF immunosensor and conventional ELISA kit (n=3). 82x80mm (600 x 600 DPI)



For TOC only 82x44mm (300 x 300 DPI)


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Fluorescence resonance energy transfer-mediated immunosensor

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