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Cascade Reaction-Mediated Assembly of Magnetic/ Silver Nano-particles for Amplified Magnetic Biosensing Yiping Chen, Yunlei Xianyu, Mingling Dong, Jiangjiang Zhang, Wenshu Zheng, Zhiyong Qian, and Xingyu Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01138 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Analytical Chemistry

Cascade Reaction-Mediated Assembly of Magnetic/Silver Nanoparticles for Amplified Magnetic Biosensing ∥

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Yiping Chen†, , Yunlei Xianyu†, , Mingling Dong†,§, , Jiangjiang Zhang†, Wenshu Zheng†, Zhiyong Qian*,§, and Xingyu Jiang*,†, ‡ † Beijing

Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, No. 11 Zhongguancun Beiyitiao, Beijing 100190, P. R. China ‡ The University of Chinese Academy of Sciences, 19 A Yuquan Road, Shijingshan District, Beijing, 100049, P. R. China § State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center. Chengdu, Sichuan, 610041, P. R. China E. mail: [email protected] (ZY Qian), phone number: (86)28-85501986; E. mail: [email protected] (XY Jiang), phone number: (86)10 8254 5558. ABSTRACT: Conventional magnetic relaxation switching (MRS) sensor suffers from its relatively low sensitivity when it comes to the analysis of trace small molecules in complicated samples. To meet this challenge, we develop a cascade reaction-mediated magnetic relaxation switching (CR-MRS) sensor, based on the assembly of silver nanoparticles (Ag NPs) and magnetic nanoparticles (MNPs) to improve the sensitivity of conventional MRS. The cascade reaction triggered by alkaline phosphatase generates ascorbic acid, which reduces Ag+ to Ag NPs that can assemble the initially dispersed MNPs to form magnetic/silver nano-assemblies, thus modulating the state of MNPs to result in the change of transverse relaxation time. The formed magnetic/silver nano-assemblies can greatly enhance the state change of MNPs (from dispersed to aggregated) and dramatically improve the sensitivity of traditional MRS sensor, which makes this CR-MRS sensor a promising platform for highly sensitive detection of small molecules in complicated samples.

The development of nanotechnology has advanced the field of biochemical analysis1-9, especially the magnetic relaxation switching (MRS) assays based on magnetic nanoparticles (MNPs)10-13. MRS allows one-step detection of analytes in opaque samples because it is homogenous and independent of optical signals that are free of both light-based interferences and multiple-washing steps14,15. However, conventional MRS assays still have some challenges16,17, particularly for the detection of small molecules through the antibody-antigen reaction18,19. The magnetic signal (transverse relaxation time, T2) in MRS depends on the state change of MNPs, where the T2 signal changes little at a low concentration of small molecules20. Consequently, it results in the low sensitivity of conventional MRS considering that small molecules only have single antigen recognition epitope and can hardly change the state of MNPs in the immunoreaction. It needs additional signal amplification steps to improve the sensitivity of conventional MRS21,22. Researchers have been improving the sensitivity of conventional MRS18,20, by integrating the magnetic separation into MRS23 to improve the sensitivity, or by using signal amplification steps including the streptavidin-biotin recognition, bioorthogonal reaction or nanoparticles to improve the binding amount of MNPs to the surface of target 19, 10, 24. The sensitivity of these developed MRS assays could be improved by one or two orders of magni-

tude, however, they still cannot satisfy the requirement for detection of trace small targets. One way to meet the challenge of conventional MRS and broaden their applications for detecting small molecules is to introduce an effective signal amplification strategy to enhance the sensitivity. Previous works prove that enzyme-mediated cascade reaction can control the growth of NPs or change the state of NPs in the redox reaction25,26. Enzyme-mediated cascade reaction can be used for signal amplification for biochemical analysis due to the high catalytic efficiency of enzymes6,27. The introduction of enzyme-mediated cascade reaction is expected to enhance the state change of MNPs in solution, so as to improve the sensitivity of conventional MRS, particularly for the detection of small molecules. In this study, we develop a cascade reaction-mediated assembly of magnetic/silver nanoparticles for MRS sensing (CR-MRS), and employ this CR-MRS sensor for highly sensitive detection of chloramphenicol (CAP), a prohibited antibiotics in milk samples. Alkaline phosphatase (ALP), a widely used enzyme, can catalyse reactions that assist the generation of Ag NPs to assemble the MNPs for enhanced T2 signals for readout. Specifically, ALP enables the removal of phosphate groups from non-reducing ascorbic acid-phosphate, and yields the ascorbic acid as a reductant to reduce Ag+ to silver NPs (Ag NPs). In this cascade reaction, the generated Ag NPs can assemble the

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dispersed carboxyl modified MNP30 (COOH-MNP30) and form the Ag NPs-MNP30 assembly NPs, resulting in the change of T2 (∆T2) signal of protons of surrounding water molecules (Scheme 1). As the Ag NPs derive from the ALP-catalyzed reactions and can further recruit MNPs for enhanced ∆T2, this approach can detect small molecules in a quantitative way such as the CAP quantification in milk samples. This CR-MRS combines the high efficiency of enzyme-mediated signal amplification and the NPs-trigged effective signal transformation and readout, which is expected to greatly improve the sensitivity of conventional MRS.

(pH=7.4, 0.01M), and the final concentration of Ab1 was 1 mg/mL. The biotin was mixed with Ab1 at a molar ratio of 20:1 (biotin to Ab1), and the mixture solution was shaken up for 1 h at room temperature. This mixture solution was transfered into a clean centrifugal ultra-filtration unit (10 KDa filter) and centrifuged at 10000 rpm for 5 min at 4 °C to remove excess sulfo-NHS-LC-biotin and ions. After three washing steps using PBS solution (pH=7.4, 0.01M), the sulfo-NHS-LC-biotin-Ab1 conjugate was collected and was diluted using PBS. Finally, this conjugate was stored this conjugate at -20 °C. Preparation of MNP30-Ab1 and MNP30-BSA-CAP conjugate 1 mg of suspend MNP30 was added into 100 µL of activated buffer (80 nM MES, pH=6.0). 10 µL of EDC (10 mg mL-1) and 10 µL of NHS (10 mg mL-1) were transferred into to the MNP30 solution. After activation for about 30 min, 1000 µL of coupling buffer PBS (pH=7.4, 0.01M) was added into the activated MNP30 solution, the above mixture solution was equally divided to two parts. After that, 0.02 mg of Ab1 or 0.01 mg of BSA-CAP conjugate was added into the activated MNP30. The mixture solution was gently stired for 2 h at room temperature, and 200 µL of 3% BSA solution was added for 0.5 h. The MNP30-Ab1 conjugate or MNP30BSA-CAP conjugate was magnetically separated from the free Ab1 or BSA-CAP conjugate by SuperMag separator at 4 °C for 24 h, and this conjugate was re-suspend using 1000 µL of PBST, and the MNP30-Ab1 or MNP30-BSA-CAP conjugate was also separated in the magnetic field, and this magnetic separation was repeated for three times. Finally, the MNP30-Ab1 or MNP30-BSA-CAP conjugate was re-suspend using PBS solution (pH=7.4, 0.01M, 0.01% BSA) and the conjugate was stored at 4 °C for further use. The CR-MRS for the detection of CAP. The coating antigen (BSA-CAP conjugate) was diluted to 50 ng/mL using the carbonate buffer (0.2 M, pH=9.6), and 100 µL of this BSA-CAP solution was transfered into 96-well plate and was incubated for 12 h at 4 oC, followed by three washing steps with PBST. 150 µL of blocking buffer (PBS solution with 3% BSA) was added to each well for 2 h. After three washing steps, 50 µL of different concentrations of CAP (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 and 500 ng/mL), 50 µL of biotin-Ab1 and 50 µL of SA-ALP (1 µg/mL) were added into each well, and were incubated for 1 h at 37 oC. After washing three times, 100 µL of 2-phospho-L-ascorbic acid trisodium salt (10 mM) was added into the well and was incubated for 0.5 h at 37 oC. 80 µL of supernatant was added into 120 µL of the mixture solution of Ag(NH3)2OH (60 µL) and MNP30(60 µL) for 2 min. The NMR analyzer was used to collect the T2 signal. The Carr-Purcell-Meiboom-Gill pulse sequences was employed to obtain T2 signal with the following parameters: the NMR frequency: 62.16 MHz; 90° pulse width: 40 µs; 180° pulse width: 80 µs; repetition time: 10 s; echo number: 600; echo spacing: 15 ms; number of scans: 1. The limit of detection (LOD) is defined as follows: LOD=3S/M, where S is the value of the standard deviation of blank samples; M is the slope of standard curve within a low concentration range. The conventional MRS for detection of CAP. 50 µL of MNP30-Ab1 solution (0.5 µg/mL), 50 µL of MNP30-BSACAP solution (0.2 µg/mL) and 100 µL of different concentrations of CAP were transfered into the bottom of the 96well microplate, and the final concentrations of CAP were 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 and 500 ng/mL. Each mixture solution was gently shaken for 30 min. 20 µL of the above mixture solution was taken out and was measured by

Scheme 1. The schematic diagram of CR-MRS assay based on ALP-triggered generation of Ag NPs for assembly of MNPs. (A) The proposed mechanism of the formation of nano-assemblies. Ascorbic acid can reduce Ag+ absorbed on the COOH-MNP30 to Ag NPs. After the nucleation and growth of Ag NPs, the dispersed COOH-MNP30 can be assembled on the Ag NPs to form Ag NPs-MNP30 nanoassemblies. (B)The principle of ALP-mediated cascade reaction for detection of chloramphenicol via ∆T2. ALPconjugated antibodies can efficiently catalyze the hydrolysis of ascorbic acid -phosphate into ascorbic acid, which results in the formation of Ag NPs-MNP30 nano-assemblies. It leads to the state change of COOH-MNP30 from dispersed to aggregated, resulting in the changes of T2 for signal readout. The amount of target determines the concentration of ALP in the immunoassay, which can be used for the quantitative determination of targets.

EXPERIMENTAL SECTION Preparation of Ag(NH3)2OH solution. 200 µL of NH3.H2O (15 M) was added into 6 mL of AgNO3 (0.1 M) , and the mixture solution was stired until the brown precipitate just dissolves. Then 3 mL of KOH (0.8 M) solution was added, and the brown precipitate reforms. After that, 190 µL of NH3.H2O (15 M) was added to the above mixture solution again to dissolve the precipitate. Finally, deionized water was added to reach a final volume of 25 mL, and the Ag(NH3)2OH solution was stored in the dark at 4 oC. Synthesis of sulfo-NHS-LC-biotin-Ab1 conjugate. The sulfo-NHS-LC-biotin was diluted using 100 µL of DMF (1 mg/mL). Ab1 solution was diluted by the PBS solution


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Analytical Chemistry NPs form and they might further interact with MNP30 to form the Ag NPs-MNP30 assembly NPs that slightly alters the absorption band of pure Ag NPs (Figure 1C). We employ X-ray photoelectron spectroscopy (XPS) to confirm the change of valence state of Ag in the ascorbic acid-mediated redox reaction. After addition of ascorbic acid into Tollens’ reagent, Ag in the mixture solution displays mainly as Ag0 (Ag NPs) and Ag+ (Tollens’ reagent), associated with the Ag3d5/2 signals at ca. 368.2 eV and 367.6 eV, respectively (Figure 1D). The XPS characterization demonstrates the redox reaction can convert Ag ions into elemental Ag NPs.

the NMR analyzer to obtain the T2 value, and each point was measured for three times (n = 3). Milk samples analysis. The blank CAP-negative milk samples were provided by the Chinese Academy of Inspection and Quarantine (Beijing, China). 9 mL of PBS solution was added into 1 mL of blank milk samples to obtain the milk matrix. To prepare a series of CAP concentrations in milk samples, a series of CAP standard solutions were prepared by dissolving CAP in the milk matrix. The solution was further diluted with the milk matrix to 0.1, 0.5, 1, 5 and 10 ng/mL. 12 blind samples were provided from the Chinese Academy of Inspection and Quarantine, and the concentrations of CAP in these samples were previously detected by HPLC-MS according to the PRC National Standard (GB 29688-2013). Each sample was assayed thrice (n=3).

RESULTS AND DISCUSSION Formation of Ag NPs-MNP30 assembly NPs. The formation of the Ag NPs-MNP30 assembly in the ALPmediated redox reaction is proposed to be a seedmediated growth model that includes two steps28,29: (1) in the nucleation step, Ag+ can be converted into small Ag seeds in the presence of ascorbic acid; (2) in the growth stage, small Ag seeds grow into large Ag NPs and finally form Ag NPs-MNP30 assembly NPs (Scheme 1A). The pH of the Ag(NH3)2OH solution is about 8.0, so the carboxyl group on the surface of MNP30 will deprotonate to be negatively charged such that it attracts lots of Ag+ ions as a result of the electrostatic interaction, facilitating the reduction reaction between Ag+ and ascorbic acid30. Ascorbic acid can reduce Ag+ ion to form small Ag NPs, and lots of small Ag NPs adsorb on the surface of COOH-MNP30 and quickly forms the heterostructured Ag NPs-MNP30 assembly NPs (Scheme 1 A). To confirm this hypothesis, we monitor the UV-vis spectrum of Ag NPs-MNP30 assembly NPs at different time points. We find that the absorbance at 420 nm dramatically increases from 0 to 10 s, and saturates at 40 s (Figure 1A), suggesting the initial formation of Ag NPs and further the assembly of Ag NPs-MNP30 NPs. We also study the change of the magnetic signal (T2) during this process. The initial formation of Ag NPs would not result in the change of T2 and only the assembly of dispersed MNP30 into aggregated MNP30 by the generated Ag NPs leads to the increase of the ∆T2 value. As expected, we find that the state change of MNP30 results in the increased ∆T2 value. We find that the increase of ∆T2 value lasts till 90 s (Figure 1B), longer than that of absorbance which suggests that the generated Ag NPs have an affinity with COOH-MNP30 to change the state of MNP30 and thus an increase in the ∆T2 value. Characterization of Ag NPs-MNP30 assembly NPs. We use UV-vis spectrum to demonstrate the formation of Ag NPs-MNP30 assembly NPs. The UV-vis spectrum of Ag+ solution exhibits an absorbance at 420 nm after the addition of ascorbic acid solution (Figure 1C), which suggests the formation of Ag NPs. Meanwhile, the UV-vis spectrum of Ag+ solution exhibits an absorption at 415 nm after the addition of ascorbic acid and COOH-MNP30 which has no characteristic absorption from 300 nm to 700 nm (Figure 1C), suggesting that Ag

Figure 1. Mechanism study of the formation of Ag NPsMNP30 assembly. (A) Time-dependent absorbance at 420 nm of Ag+-MNP30 solution after the addition of 0.01 mM ascorbic acid. (B) Time-dependent ∆T2 value of Ag+MNP30 solution after the addition of 0.01 mM ascorbic acid. (C) UV-vis spectra of MNP30, Ag NPs and Ag NPsMNP30 assembly NPs. (D) XPS spectra of Ag3d5/2 in the Ag NPs-MNP30 assembly NPs. The blue line represents the Ag0 (Ag NPs), and the red line represents the Ag+ ion. We employ transmission electron microscope (TEM) to characterize and validate the formation of Ag NPsMNP30 assembly. The size of the dispersed COOHMNP30 is about 30 nm (Figure 2A), and the energydispersive X-ray spectroscopy (EDS) indicates the existence of Fe in the COOH-MNP30 (Figure 2B). In the presence of COOH-MNP30, the solution generates Ag NPs with a regular shape because the carboxyl group can modulate the shape of Ag NPs in alkaline environment31(Figure 2C). The size of the generated Ag NPs is about 100 nm, and the COOH-MNP30 assembled around Ag NPs to form the Ag NPs-MNP30 assembly NPs, confirming the state change of MNP30. Elemental analysis by EDS shows the co-existence of both Fe and Ag that indicates the formation of the satellite structure-like Ag NPsMNP30 assembly (Figure 2D). The reason why a satellite structure-like rather than a core-shell structure forms in the assembly is due to the lattice mismatch between Ag and Fe, given that the lattice constant of Ag and Fe is 4.09 and 2.8728, respectively. Our previous study shows that the Au@Ag core-shell structures form when Au NPs are used as the substrate in the ascorbic acid and Ag+-



mediated redox reaction32 because Ag and Au have similar lattice constants, and the lattice constant of Au and Ag is 4.08 and 4.0928, respectively. Different from Au NPs, the newly deposited Ag atoms tend to grow epitaxially on the MNP30 template when Fe-containing MNP30 is used as the substrate. Consequently, the satellite structure-like Ag NPs-MNP30 assembly rather than the coreshell assembly forms after the ascorbic acid and Ag+mediated reaction.

Surface chemistry of MNPs. The surface chemistry of MNPs affects the growth of Ag NPs in the redox reaction28. We employ UV-vis spectrum to characterize the generated Ag NPs. The formed Ag NPs have a distinct absorption at 420 nm when COOH-MNP30 is used, while the absorption at 420 nm is not significant when NH2MNP30 is used (Figure 3A), showing that the COOHMNP30 contributes to the formation of Ag NPs. The dynamic light scattering (DLS) results show that the size of single MNP30, NH2-MNP30-Ag NPs and COOH-MNP30Ag NPs is about 30 nm, 80 nm and 120 nm, respectively (Figure 3B), which further suggest that the surface chemistry of MNPs affect the formation of MNPs-Ag NPs. The surface ligand of the nanoparticles is important to control the growth of NPs in the seed-mediated growth process33. For example, our previous work demonstrates that the electrostatic interaction between the substrate (Au NPs) and Ag+ is crucially important in the formation of Ag NPs30. The positively charged Au NPs, such as cetyltrimethyl ammonium bromide or quaternary ammonium-capped Au NRs are not able to recruit Ag+ for the nucleation and further growth of Ag NPs. In contrast, the negatively charged Au NPs (such as polystyrene sulfonate-coated Au NRs) can efficiently absorb Ag+ to increase the local concentration for the nucleation and generation of Ag NPs. In this study, we also found that the structure of generated Ag NPs is irregular when NH2MNP30 is used as the substrate, and most NH2-MNP30 does not absorb on the surface of Ag NPs (Figure 3C). Ag NPs tend to nucleate and grow individually in the solution, as demonstrated by EDS (Figure 3D). These results show that COOH-MNP30 plays an important role in controlling the growth of Ag NPs, which conforms to related work28. The surface ligand mainly affects the surface charge of MNP30 that leads to the difference in recruiting Ag+ for the formation of Ag NPs. We study the zeta potential of COOH-MNP30, Ag NPs, Ag NPs-MNP30, and NH2-MNP30 (Figure 3E). The zeta potential of NH2MNP30 is about 26 mV, while that of COOH-MNP30 is about -28 mV. The difference in the surface potential influences the electrostatic interaction between Ag+ and the functionalized groups in the MNPs and further dictates the generation of Ag NPs. Specifically, the negatively charged COOH-MNP30 will enrich Ag+ by electrostatic interactions in this redox reaction, resulting in a high local concentration of Ag+ around COOH-MNP30 that is vital to the nucleation and seed-mediated growth. These results show that the surface chemistry of MNP30 plays an important role in controlling the growth of Ag NPs. Ag+ can bind to COOH-MNP30 via electrostatic attraction may affect the colloidal stability of MNP, thus we investigate this possible effect by two experiments. We firstly investigated the T2 value of MNP30 solution before and after addition of Ag+ (Figure S2 A). We also characterize the size change of MNP30 before and after addition of Ag+ using DLS (Figure S2B). These results both suggest that the interaction between Ag+ and COOH-MNP30 via electrostatic attraction do not affect the colloidal stability of COOH-MNP30. We further demonstrate that the surface ligand of MNP30 affects the ALP sensing by using COOH-MNP30

Figure 2. Characterization of MNP30, Ag NPs, and Ag NPs-MNP30 assembly NPs by transmission electron microscope (TEM) and energy-dispersive X-ray spectroscopy (EDS), respectively. The TEM images of (A) MNP30, (C) Ag NPs-MNP30 assembly NPs, and (E) Ag NPs, and ESD spectra of (B) MNP30, (D) Ag NPs-MNP30 assembly NPs and (F) Ag NPs. For comparison, we also characterize the Ag NPs without the use of COOH-MNP30. The structure of generated Ag NPs is irregular (Figure 2E), and EDS proves that Ag+ can be converted into Ag NPs in the redox reaction (Figure 2F). In the absence of COOH-MNP30, Ag+ ions are uniformly distributed in the solution, and the generated Ag NPs easily aggregate to form large clusters due to the high specific surface energy which results in the connective and irregular Ag NPs (Figure 2E). In contrast, the negatively charged COOH-MNP30 enriches Ag+ by the electrostatic interactions which increases the local concentration of Ag+ that is beneficial for the nucleation and growth of Ag NPs. The generated Ag NPs are individual with regular shapes that facilitate the formation of the COOH-MNP30-Ag NPs assembly (Figure S1). These results indicate that MNP30 can affect the growth of Ag NPs which further recruit MNP30 to form the hybrid NPs.


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Analytical Chemistry and NH2-MNP30 as substrates. As the generation of Ag NPs and thus the aggregation of MNPs are quantitatively dependent on the ALP-catalyzed cascade reactions, the T2 signal can serve as the quantification of ALP in the assay. The ∆T2 increases when the concentration of ALP is from 0 to 150 U/L and the COOH-MNP30 is used (Figure 3F). Because the generated Ag NPs have characteristic surface plasmon resonance32, we found that the color of the mixture solution changes from colorless to pale yellow, deep yellow and dark brown when the concentration of ALP is from 0 to 150 U/L (Figure S3). Compared with COOH-MNP30, the ∆T2 value has a negligible change when the concentration of ALP increases from 0 to 150 U/L and the NH2-MNP30 is used (Figure 3F), suggesting that the surface chemistry of MNPs is of great significance in the CR-MRS. The carboxyl group in the MNPs facilitates the generation of Ag NPs that further help the assembly of MNPs, while the amino group in the MNPs helps little with the formation of Ag NPs-MNP30 assembly NPs, which further proves the importance of the surface chemistry of MNP30 in this assay.

detection by using both the generated Ag NPs for the optical signal readout and the Ag NPs-MNP30 assembly for the magnetic signal readout. In the Ag+-mediated redox reaction, the solution of Ag NPs-MNP30 assembly shows a yellow color because the generated Ag NPs have a characteristic surface plasmon resonance. The ∆T2 resulted from Ag NPs-MNP30 assembly is quantitatively dependent on the concentration of ALP which can be used as the magnetic readout (Figure S4). The limit of detection (LOD) for detection of ALP is 1.0 U/L when the absorbance of Ag NPs is employed for the readout. In contrast, the LOD for detection of ALP using T2-based readout is 0.2 U/L, which suggests that the T2 signal is a more sensitive readout in this ALP-mediated redox reaction. We optimize the conditions of the cascade reaction to obtain the best sensitivity of CR-MRS, including the concentrations of MNP30, ascorbic acid-phosphate and Ag+, the pH value of the ALP-mediated enzymatic reaction. The optimized conditions are 0.1 µg/mL of MNP30, 16 mM of ascorbic acid-phosphate, 2.4 mM of Ag+ and NaHCO3-Na2CO3 solution (pH=9.6) (Figure S5). Under the above optimized conditions, we first compare the CRMRS with conventional MRS for the detection of CAP. CAP is a widely used antibiotic against bacterial infections twenty years ago, but it is prohibited in agriculture due to its high toxicity to humans34-37. The abuse of CAP still occurs even though many countries have established a zero-tolerance policy. To achieve highly sensitive detection of trace CAP in agricultural samples is urgently needed. In the CR-MRS, the concentration of CAP determines the amount of generated Ag NPs, which results in the state change of COOH-MNP3o NPs and the ∆T2. The ∆T2 increases as the concentration of CAP changes from 0.01 ng/mL to 500 ng/mL (Figure 4B), with a linear relationship falling in the range of 1 to 200 ng/mL. The linear equation is Y=77.6X+23.9 (X=lg[CAP (ng/mL)],R2=0.98) (Figure 4C), and the LOD of CRMRS for detection of CAP is 0.015 ng/mL (LOD=3S/M). The selectivity test of the CR-MRS for CAP detection shows that the ∆T2 value from CAP measurement is much higher than that from other analogues (Figure 4D), suggesting that this strategy has a good specificity due to the high specificity of the immuno-recognition. Comparison with conventional MRS and enzyme-linked immunosorbent assay (ELISA) for CAP detection. In conventional MRS, the aggregation of MNPs relies on the immuno-recognition between MNP30-BSA-CAP conjugate and MNP30-antibody conjugate (Figure 4A). The aggregation of MNPs in conventional MRS is limited because monoclonal antibody has only single valency to recognize the targets. Without effective signal transformation and amplification in conventional MRS, the sensitivity for detection of small molecules is low. The linear range of the conventional MRS for the detection of CAP is 10-200 ng/mL and the LOD (3S/M) is 0.65 ng/mL, respectively (Figure 4C). Compared with conventional MRS, the sensitivity of CRMRS for CAP detection has been improved by 50 folds and the linear range is also improved by one order of magnitude. Compared with previous work that employs biotin-streptavidin to enhance the aggregation of MNPs

Figure 3. Characterizations of MNP30, Ag NPs, and Ag NPs-MNP30 assembly NPs. (A) UV-vis spectra of COOHMNP30-Ag NPs assembly NPs and NH2 -MNP30-Ag NPs assembly NPs in the redox reaction. (B) DLS analyses of NH2-MNP30-Ag NPs assembly, COOH-MNP30-AgNPs assembly and MNP30. (C) TEM image of NH2-MNP30-Ag NPs assembly. (D) EDS spectrum of small Ag NPs. (E) Zeta potentials of COOH-MNP30, Ag NPs, COOHMNP30-Ag NPs assembly and NH2-MNP30. (F) The relationship between T2 signal and the concentrations of ALP in the COOH-MNP30 and NH2-MNP30-mediated redox reaction. The sensitivity and selectivity of CR-MRS for detection of CAP. We compare the sensitivity of ALP



to improve the sensitivity of conventional MRS for detection of small molecules19, this CR-MRS is superior in the signal amplification. The streptavidin only has 4 binding sites for biotin to enhance the aggregation of MNPs, thus one streptavidin can only result in the aggregation of four MNPs. In contrast, one Ag NPs (100 nm-120 nm) can bind about 64 COOH-MNP30 under the optimized condition, and ALP can effectively covert Ag+ to Ag NPs that contribute to the high sensitivity of CR-MRS. In addition, conventional MRS requires the conjugation of antibody on the MNP30 to prepare MNP30-antibody conjugate which affects the suspension stability of MNP30, and thus the stability of MRS sensor. The CR-MRS is conjugation-free which can greatly retain the suspension stability of MNP30 and ensure the stability of the assays. We also employed ELISA for detection of CAP. ELISA is a widely used method for detection of veterinary drug residues in food safety. The LOD of ELISA for detection of CAP is 0.056 ng/mL(Figure S6A) and the linear range of ELISA for detection of CAP is 0.5-20 ng/mL(Figure S6B). The result shows that the analytical performances of CR-MRS are better than those of ELISA for detection of CAP.

is under 18.5%, suggesting the good stability of CR-MRS. In contrast, traditional MRS cannot detect the spiked samples of 0.1 ng/mL and 0.5 ng/mL due to its low sensitivity. The main contents inside milk include fat, protein, vitamin and so on. Fat and protein affect little on the magnetic signal. Vitamin, or other reducing agents in milk may potentially interfere with the formation of magnetic/silver nano-assemblies induced by the cascade reactions. However, the washing steps in this CR-MRS can avoid this problem because it can effectively wash away the reducing agents to reduce the interference. In contrast, in conventional fluorescence-based assay, proteins may absorb on the well plate that will affect the optical signal. We detect the concentrations of CAP in 12 real milk samples, and the CAP levels in the real milk samples are pre-determined by high performance liquid chromatography-mass spectrum (HPLC-MS)38, which is a gold standard for detection of trace antibiotics in food samples. Sample 3, 4, 7 and 8 are detected to be CAP-positive by both CR-MRS and HPLC-MS, and other samples are

Figure 4. The sensitivity and selectivity of CR-MRS and conventional MRS for detection of CAP. (A) The schematic diagram of conventional MRS for detection of CAP. (B) The standard curve of CR-MRS and conventional MRS for detection of CAP. The concentration of CAP ranges from 0.001 ng/mL to 500 ng/mL. (C) The linear range of CR-MRS and conventional MRS for CAP detection (CAP ranging from 1 to 200 ng/mL). (D) The specificity of CR-MRS for detection of CAP. The concentration of CAP is 50 ng/mL, and the concentration of other analogues is 500 ng/mL.

Figure 5. The results of CR-MRS, conventional MRS and HPLC-MS for detection of CAP in real milk samples. (A) The results of CR-MRS, conventional MRS and HPLC-MS for detection of CAP in real milk samples; (B) The comparison of CAP levels measured by the CR-MRS and HPLC-MS, showing a good consistency between two methods. The milk samples are from Chinese Academy of Inspection and Quarantine (Beijing, China), and the concentration of CAP in these samples are determined by HPLC-MS.

Real sample analysis. To demonstrate the application of the CR-MRS, we employ CR-MRS and conventional MRS for detecting CAP in 8 spiked milk samples to investigate its recovery in complex matrix. The CRMRS can detect the CAP levels of spiked samples (0.1, 0.5, 1, 5, and 10 ng/mL) with acceptable recoveries (63%-106.3%) (Table S1). In addition, the coefficient of variation (CV) at different spiked concentrations of CAP


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Analytical Chemistry detected to be CAP-negative (Figure 5A). More importantly, the quantitative results of this CR-MRS for detection of CAP in these CAP-positive milk samples agree well with those of HPLC-MS (Figure 5B and Table S2). In contrast, only sample 8 is detected to be CAPpositive by MRS, suggesting that it has insufficient sensitivity (Figure 5A). Although HPLC-MS is a gold standard method for detection of antibiotic residues in food samples because of its high sensitivity and accuracy, it needs complex sample pre-treatment and high expense. In contrast, the CR-MRS is a straightforward and rapid method that only needs simple sample pre-treatment, and has great potential in detection of trace hazardous substance in food safety. We have compared the analytical performance of the CR-MRS with current immunoassays39-42 for CAP detection in terms of the LOD, the detection range, and operability (Table S3). Many previous works for detection of CAP have good sensitivity, and the LOD can reach about 0.001-0.1 ng/mL, which can satisfy the requirement for detection of CAP. However, these methods usually need the complex nanoparticles-mediated signal amplification system43,44 which requires laborious work on the preparation of nanoparticles. In contrast, CR-MRS employs a commercial enzyme to amplify the magnetic signal which is an effective and straightforward signal amplification strategy.

formance of the CR-MRS with the current immunoassays for CAP detection. Additional analytical data, including Figure S1-S4 Table S1-S3

AUTHOR INFORMATION Corresponding Author [email protected] (ZY Qian) [email protected] (XY Jiang)

Author Contributions ∥

These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank the National Natural Science Foundation of China (81671784, 21505027, 81361140345, 21535001, 81730051, 21761142006), Beijing Nova Program (Z181100006218017), the Ministry of Science and Technology of China (2013YQ190467), Chinese Academy of Sciences (XDA09030305, 121D11KYSB20170026), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2018047) for financial support.

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In conclusion, we employ a cascade reaction-mediated assembly of magnetic/silver nanoparticles for amplified sensitivity of conventional MRS.The CR-MRS is a promising magnetic sensor for detection of small molecules because of its high sensitivity and convenient operation. Although current CR-MRS developed in this work is incapable of large-scale detection of samples, we will focus on developing the high-throughput CR-MRS combined with microfluidic technology to improve the efficiency of analysis in further work. Besides analytics of small molecules, this enzyme-mediated assembly strategy has great potential for in vivo imaging and diagnosis because many enzymes are important cellular biomarkers and the magnetic signal has good penetrability for in vivo sensing. We hope this ALP-mediated controlled assembly strategy can be further developed for in vivo biosensing applications.

ASSOCIATED CONTENT Supporting Information Materials and equipment and additional analytical data. Additional experimental data, including the color change of COOH-MNP30 in the Tollens' reagent with the increasing concentrations of ALP, the TEM images of generated Ag NPs-MNP30 assembly in the ALP-mediated redox reaction, the result of optical readout based on the color change of generated Ag NPs and the T1 assay based on Ag NPs-MNPs assembly for detection of ALP, optimization of conditions of CR-MRS, the results of detection of spiked CAP in blank samples by CR-MRS and conventional MRS(n=3), the results of detection of CAP in real milk samples by CR-MRS and HPLC-MS (n=3), the comparison of the analytical per-



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For TOC use only


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