T1-Mediated Nanosensor for Immunoassay Based on an Activatable

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A T-Mediated Nanosensor for Immunoassay Based on Activatable MnO Nano-Assembly 2

Zixin Liu, Yunlei Xianyu, Wenshu Zheng, Jiangjiang Zhang, Yunjing Luo, Yiping Chen, Mingling Dong, Jing Wu, and Xingyu Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04817 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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Analytical Chemistry

A T1-Mediated Nanosensor for Immunoassay Based on Activatable MnO2 Nano-Assembly , ,

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Zixin Liu † ‡ #, Yunlei Xianyu ‡ #, Wenshu Zheng ‡ #, Jiangjiang Zhang ‡, Yunjing Luo * †, Yiping Chen *,‡, Mingling Dong ‡, Jing Wu ‡ and Xingyu Jiang*,‡,§ †

College of Life Science and Bioengineering, Beijing University of Technology, No.100, PingLeYuan, ChaoYang District, Beijing, 100124, P.R. China. ‡ Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nano-safety, CAS Center for Excellence in Nanoscience. National Center for NanoScience and Technology. 11 BeiYiTiao, ZhongGuanCun, Beijing 100190, P.R. China. § The University of Chinese Academy of Sciences, 19 A YuQuan Road, ShiJingShan District, Beijing, 100049, P. R. China. Corresponding Authors E. mail: [email protected] (YJ Luo), phone number: (86)10 67396211; E. mail: [email protected](YP Chen), phone number: (86)10 82545631; E. mail: [email protected] (XY Jiang), phone number: (86)10 8254 5558 ABSTRACT: Current magnetic relaxation switching (MRS) sensors for detection of trace targets in complex samples still suffer from limitations in terms of relatively low sensitivity and poor stability. To meet this challenge, we develop a longitudinal relaxation time (T1)-based nanosensor by using Mn2+ released from the reduction of MnO2 nano-assembly that can induce the change of T1, thus can greatly improve the sensitivity and overcome the “hook effect” of conventional MRS. Through the specific interaction between antigen and antibody-functionalized MnO2 nano-assembly, the T1 signal of Mn2+ released from the nano-assembly is quantitatively determined by the antigen, which allows for highly sensitive and straightforward detection of targets. This approach broadens the applicability of magnetic biosensors, and has great potential for applications in early diagnosis of disease biomarkers.

Highly sensitive and reliable detection of low concentration of targets in complex samples has become increasingly important in not only scientific research 1-3 but also clinical diagnosis4-6, which leads to the development of nanoparticles-mediated analytical methods7,8. Nanoparticles have been widely used for signal readout9,10 or signal amplification11,12 that greatly improved the analytical performance13,14(sensitivity and stability) due to their excellent magnetic15, optical16 or electrical properties17. Compared with optical or electrochemical sensors, magnetic nanoparticles (MNPs)-mediated magnetic sensors have received increasing attention in biochemical analysis due to their excellent anti-interference properties18,19. Most complex samples have negligible magnetic signals20, so the magnetic biosensors require simple or even no sample pre-treatment for detection of biomarkers in complex samples. Among the magnetic sensors, magnetic relaxation switching (MRS) sensing using the transverse relaxation time (T2) as signal readout is classic21, which has been used to detect a range of targets from small molecules to

cancer cells due to its straightforward operation and rapid response22,23. However, the biggest challenge of conventional MRS is its relatively low sensitivity and it cannot detect targets of low abundance because conventional MRS lacks effective signal amplification. In conventional MRS, it is the target that induces the state change of MNPs (from dispersion to aggregation), resulting in the T2 change of proton of surrounding water molecules20,24. Nevertheless, the target can only bind limited number of MNPs and the detection signal cannot be effectively amplified to a high degree that rarely exceeds a few hundreds folds25. Though many efforts have been made to improve the sensitivity of conventional MRS, such as using the layer-by-layer bio-conjugation strategy26, the amplification effect is still limited owing to the steric hindrance of MNPs. In conventional MRS, Fe3O4/Fe2O3 NPs have been widely used as magnetic signal probes27-29. Recent studies reported that manganese (Mn)-doped ferrite (MnFe2O4) particles have higher magnetization than Fe3O430,31, which holds potential for improving the sensitivity of traditional

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Fe-based MRS sensors. In addition, Mn-containing nanoparticles such as MnO2 nanoparticles have been used in magnetic resonance imaging32-34. These studies suggest that Mn-containing nanoparticles may have better performance than Fe-containing nanoparticles as the magnetic probe for sensing applications35. Mn has a variety of valence states, for instance, Mn2+ is a paramagnetic ion that has five unpaired electrons, a long electronic relaxation time, and labile water exchange35,36. Under the same conditions, Mn2+ has greater longitudinal relaxivity than that of Fe3+ in aqueous solution, making it a more attractive candidate for magnetic sensing37,38.

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mediated formation of MnO2 nano-assembly can improve the loading of BSA-MnO2 NPs on the surface of targets. In the second round, lots of Mn2+ can be released from the MnO2 nano-assembly after the reduction reaction by AA. Since previous studies demonstrated that the longitudinal relaxivity of Mn2+ is higher than that of Mn atoms in the MnO2 nanostructure33, the conversion of Mn-containing nanostructure into Mn2+ can effectively amplify the signal for magnetic readout. Mn2+ released from the MnO2 nanoassembly has outstanding ability to produce T1 signal, and the detectable concentration of Mn2+ can reach nanomole (nM) level when using T1 for the signal readout. Since the number of MnO2 nano-assembly that bind to the magnetic beads (MBs)-antibody (MBs-Ab1) after magnetic separation is correlated to the concentration of target, this method can be employed for the quantitative analysis of target molecules such as inflammatory biomarkers.

EXPERIMENTAL SECTION Preparation of Bull Serum Albumin (BSA)-MnO2 NPs. We prepare the BSA-MnO2 NPs refer to the previous litera42 ture with slight modification. Briefly, we dissolve 25 mg BSA in 10 mL ultrapure water. We add 50 µL of 100 mM Manganese acetate tetra-hydrate (MnAC2) aqueous solution and keep stirring for 2 min at room temperature (RT). We add 50 µL of 1 M NaOH solution and keep stirring for 7 h. After dialysis by double distilled water for 2 days, we centrifuge the solution at 14000 rpm for 30 min. Finally, we collect the BSA-MnO2 NPs and store at 4 °C for further use. Preparation of Ab2-tetrazine(Tz), BSA-MnO2-Tz, BSAMnO2-cycloolefin(TCO) conjugates. We prepare 10 mM of the Tz and TCO reagent in DMF. We mix the protein samples and Tz/TCO at a molar ratio of 1:40 (protein samples to labeling reagents), and incubate the mixture at RT for 40 min. After that, we stop the reaction by adding 20 µL of quenching buffer (500 mM of Tris-HCl, pH 8.0). After termination for 5 min, we transfer the mixture solution to a centrifugal ultrafiltration unit (10 kD filter) and centrifuge at 10000 rpm for 20 min at 4 °C for three times to remove unreacted reagent. We collect the conjugates and dilute it using PBS buffer solution and store at 4°C for further use. Preparation of MnO2 nano-assembly-Ab2 conjugates. We mix 1135 µL of 2 µg/mL BSA-MnO2-Tz and 1135 µL of 32 µg/mL BSA-MnO2-TCO and incubate them at RT for 40 min. We transfer the reaction mixture into a clean ultra-filtration unit (100 kD filter) and centrifuge at 10000 rpm for 30 min at 4 °C to remove the unbound BSA-MnO2-TCO conjugates. We collect the MnO2-TCO nano-assembly and dissolve it in PBS buffer. After that, we add 0.4 mg of Ab2-Tz and incubate the reaction at RT for 40 min. We obtain the MnO2 nanoassembly-Ab2 conjugates using the ultra-filtration unit (100 kD) and centrifuge at 10000 rpm for 30 min. We re-disperse the conjugates in PBS buffer and store them at 4 °C for further use. Preparation of MBs-Ab1 conjugates. We prepare the MBs-Ab1 conjugates using NHS/EDC amidization reaction. We suspend 5 mg of carboxyl modified MBs into 0.5 mL of

Scheme 1. The scheme of MnO2-T1 sensor for detection of target. (A) The process of preparation of Ab2-functionalized MnO2 nano-assembly by click reaction. (B) The scheme of MnO2-T1 sensor for highly sensitive and quantitative detection of targets. In the immuno-reaction, the sandwich immune-complex “MBs-target-MnO2 nano-assembly” forms. The number of MnO2 nano-assembly bound to the MBs is determined by the concentration of target in samples, which 2+ can be further converted into Mn by ascorbic acid to dramatically enhance the T1 signal for readout.

Herein, we introduce a straightforward signal generation mechanism that uses Mn2+ released from the reduction of MnO2 nano-assembly to improve the sensitivity of conventional Fe3O4 NPs-based MRS biosensors. In this strategy, we prepare Bull Serum Albumin (BSA)-capped MnO2 nanoparticles (NPs) and use the click reaction for the layer-by-layer assembly of MnO2 nano-assembly and the antibody functionalization on the MnO2 nano-assembly. When the antibody-functionalized MnO2 nano-assembly bind to the target molecules and unbound ones are removed by magnetic separation, Mn2+ can be released from the redox reaction between the MnO2 nano-assembly and ascorbic acid (AA). The released Mn2+ can lead to a dramatic enhancement in the T1 signal, and the sensitivity of this MnO2-T1 sensor is expected to be significantly improved due to the tworound amplification. In the first round, the click reaction-

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activation buffer. We transfer 80 µL of EDC (10 mg/mL) and 40 µL of NHS (10 mg/mL) to the MBs solution. After activation for 30 min, we wash the activated MBs with 0.5 mL of PBS buffer for three times, and disperse it in 0.5 mL of PBS buffer. We add 0.2 mg of Ab1 into the activated MBs, and incubate the mixture at RT for 2 h. We wash the mixture solution with 0.5 mL of PBST washing buffer for three times to remove the unbound Ab1. We add 0.6 mL of 3% BSA solution for 30 min blocking. We wash the above MBs-Ab1 conjugates with 0.5 mL of PBST washing buffer for three times, and disperse them in 0.5 mL of PBS buffer, and store the MBs-Ab1 conjugates at 4°C for further use. Preparation of MNPs-antibody (MNPs-Ab) conjugates. We transfer 0.2 mL of the MNPs into a 1.5 mL tube and add 0.2 mL of activation buffer (100 mM MES buffer, pH 4.8). We add 50 µL of EDC (4 mg/mL) and 50 µL of NHS (2 mg/mL) to the MNPs solution. After reaction at RT for 10 min with continuous mixing, we add 0.5 mL of the coupling buffer (0.1 M of PBS solution, pH=8.0) to the activated MNPs, and immediately add 0.5 mg of Ab1 or Ab2. We gently stir the mixture solution at RT for 2 h. We add 10 µL of the quenching solution, and incubate them for 10 min at RT. After that, we collect the MNPs-Ab conjugates from the unbound Ab1 or Ab2 by a magnetic separator at 4 °C for 24 h, and re-suspend the products using 1 mL of PBS buffer. We carry this magnetic separation step twice to separate the MNPs-Ab conjugates in the magnetic field. We re-suspend the conjugated MNPs with 1 mL of the washing/storage buffer and store them at 4 °C for further use. Optimized procedure for procalcitonin (PCT) detection. We choose six different concentrations of BSA-MnO2Tz (0, 1, 2, 3, 4, and 5 µg/mL) and five different concentrations of BSA-MnO2-TCO (4, 8, 16, 32, and 64 µg/mL) to synthesize MnO2 nano-assembly. We attempt six different amounts of Ab2-Tz (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg) to synthesize MnO2 nano-assembly-Ab2 conjugates. Based on the above conditions, we investigate the optimized concentration of MBs-Ab1 using different concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1 mg/mL). We investigate the concentration of AA and the time of AA-mediated reduction reaction.

guarantee the consistency of temperature. We use inversion-recovery pulse sequences for T1 measurements with the following parameters: the NMR frequency, 62.16 MHz; pulse separation, 10 ms; 90o pulse width, 32 µs; 180o pulse width, 64 µs; number of scans, 1; repetition time, 10 s. We repeat all the experiments at least three times to ensure the accuracy of the measurement. For each interval, the change in T1 (ΔT1) was calculated using the following equation: ΔT1= T1 blank- T1 sample Where T1 sample and T1 blank are the average T1 relaxation times of the triplicates of the sample and blank groups, respectively. The blank sample represents the AA aqueous solution. To obtain the limit of detection (LOD), we employ the following formula: LOD=3S/M (S: the value of the standard deviation of blank samples; M: the slope of standard curve within the low concentration range). The process of traditional T2-mediated MRS for detection of PCT. We transfer 50 µL of MNPs-Ab1 solution (1 µg/mL), 50 µL of MNPs-Ab2 solution (1 µg/mL) and 100 µL of different concentration of PCT (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 50, 100, 500, 1000, 2000, 5000 ng/mL) onto the bottom of the 96-well microplate. Each mixture solution is gently shaken at RT for 30 min. We take out 20 µL of the above mixture solution and measure T2 value by the NMR analyzer, and we obtain the average T2 value from three independently prepared samples (n = 3). Human sample analysis. We collect the PCT serum samples which are from the people who may suffer bacterial infection from Beijing Friendship Hospital (China) in accordance with the rules of the local ethical committee (2017-P2099-01).

RESULTS AND DISCUSSION The principle of MnO2-T1 sensor. We study the T1 signals of different concentrations of Mn2+ and MnO2 NPs. The ΔT1 increases when the concentration of Mn2+ is from 0 to 109.88 µg/mL, and the lowest detectable concentration of Mn2+ is 5.49 ng/mL (Figure 1A). For MnO2 NPs, the ΔT1 increases when their concentration is from 64 to 1040 µg/mL. The significant difference in the ΔT1 value makes possible the sensing strategy that uses AA to convert the MnO2 NPs into Mn2+ to amplify the T1 signal (Figure 1B). AA can partially reduce MnO2 to free Mn2+ and the lowest detectable concentration of MnO2 NPs by T1 signal is 8 µg/mL, an 8-fold higher sensitivity than that of MnO2 NPs without AA. The chemical equation is as follows:

Procedure of MnO2-T1 sensor for detection of PCT. We add 0.9 mL of different concentrations of PCT (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 50, 100, 500, 1000, 2000, 5000 ng/mL) to 0.1 mL of 0.6 mg/mL MBs-Ab1 solution, respectively, and we shake these mixture solutions at RT for 30 min. We wash the MBs with 0.5 mL of PBST washing buffer for three times, and dissolve it in 0.3 mL PBS buffer. Thereafter, we add 0.2 mL of MnO2 nanoassembly-Ab2 conjugates to the MBs solution and incubate the mixture for another 30 min. We wash the immuno-complex for three times with PBST washing buffer and disperse it in 0.1 mL of PBS buffer. We add 0.1 mL of 0.5 mM AA to reduce the MnO2 nano-assembly to Mn2+. After magnetic separation, we take out 20 µL of the above mixture solution and measure T1 value via a NMR analyzer, and we obtain the average T1 value from three independently prepared samples (n=3). Before obtaining the T1 signal, we place all the samples at 35 °C for 2 min to

In the MnO2 NPs, Mn atoms are coordinated in an octahedral geometry to six oxygen atoms which are shielded

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2+

from aqueous environment and get little contact with the protons of surrounding water molecules, leading to the low longitudinal relaxivity. In comparison, the Mn2+ released from the MnO2 NPs can directly contact with the protons of water molecules, thereby leading to a high longitudinal relaxivity39. In addition, AA can reduce MnO2 to Mn2+ that contributes to the enhancement in the T1 signal40. Both the interaction with the protons of surrounding water molecules and the alternation of valence state result in the significant change in the T1 signal. To study the principle of AA-mediated T1 signal amplification, we use 1 mM of hydrochloric acid (HCl, nonreducibility) to dissolve the BSA-MnO2 NPs, and study the change of T1 of BSA-MnO2 NPs solution before and after addition of HCl. T1 value has no significant difference (Figure S1), suggesting that non-reducing acid cannot effectively convert BSA-MnO2 NPs into Mn2+. This result also proves that the change in the valence state of Mn is a main factor that results in the change of the T1 signal. We investigate the effect of magnetic beads (MBs) to the T1 signal because AA may dissolve MBs and release Fe3+/Fe2+, which can decrease the T1 value of aqueous solution. The T1 value changes little when AA is added into the MBs solution at different time points (from 10 to 60 min) (Figure S2). In contrast, the T1 value has a significantly decrease when AA is added into the BSA-MnO2 NPs solution, suggesting that MBs has negligible effect on the T1 signal in this assay.

and NaBiO3 (a color-development reagent for Mn ) aqueous solution We filter the unreacted NaBiO3 powder before we take photo. We take the photo using a digital camera (Nikon D90, Nikon Corporation, Tokyo, Japan)

We employ the X-ray photoelectron spectroscopy (XPS) to confirm the change of the valence state of Mn in the AA-mediated redox reaction with BSA-MnO2 NPs (Figure 1C). The BSA-MnO2 NPs display two binding energy bands at 655 eV and 642 eV, due to the characteristic binding energies of Mn 2p1/2 at 655 eV and Mn 2p3/2 at 642 eV41. In contrast, after the redox reaction between AA and BSA-MnO2 NPs, the BSA-MnO2 NPs display no characteristic binding energy bands. Since the MnO2 NPs are converted into Mn2+ that is washed away, we obtain little signal of the binding energies of Mn 2p1/2 and Mn 2p3/2. The XPS characterization demonstrates the initial existence, and later disappearance of Mn in the NPs. To confirm that the change of the valence state of Mn in the AA-mediated redox reaction with BSA-MnO2 NPs, we use the sodium bismuth oxide (NaBiO3) as the indicator for the color development of Mn2+. Mn2+ can specifically react with BiO3- and produce purplish-red MnO4-, and the color of solution will change from colorless to purplish-red. AA can convert BSA-MnO2 NPs into Mn2+, which specifically reacts with NaBiO3 and produces purplish-red MnO4- (Figure 1D), further confirming that the MnO2 NPs are converted into Mn2+ in the AA-mediated redox reaction.

Figure 2. The characterization of BSA-MnO2 NPs. (A) TEM image of BSA-MnO2 NPs. (B) DLS of BSA-MnO2 NPs. (C). EDX spectra of BSA-MnO2 NPs (Element Cu occurs as a component of TEM copper grids); (D) HRTEM image of BSAMnO2 NPs.

Figure 1. Mechanism of AA-mediated T1 signal enhance2+ ment when MnO2 nano-assembly converts to Mn . (A) The 2+ relationship between the ΔT1 and the concentration of Mn ranging from 0 to 109.88 µg/mL. (B) The T1 signal enhance2+ ment of AA-mediated reduction of MnO2 NPs to Mn . The concentration of BSA-MnO2 NPs is from 0 to 1040 µg/mL. (C) High resolution Mn (2p) XPS spectra of BSA-MnO2 NPs before and after the introduction of AA. (D) The photograph of 2+ the color change resulting from the reaction between Mn

Characterization of BSA-MnO2 NPs, BSA-MnO2TCO, BSA-MnO2-Tz, antibody-Tz and MnO2 nanoassembly. We employ transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), energy dispersive X-ray detector (EDX) and dynamic light scattering (DLS) to characterize the BSA-

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MnO2 NPs. The TEM image shows that BSA-MnO2 NPs are spherical and the average size is approximately 10.2 nm (Figure 2A) and the interplanar distance is about 0.28 nm (Figure 2D). DLS shows that the size of BSA-MnO2 NPs is about 12 nm (Figure 2B). The result of EDX proves that the Mn and oxygen elements consist the BSA-MnO2 NPs (Figure 2C). Interestingly, we find that the prepared BSA-MnO2 NPs can catalyze the oxidation of tetramethylbenzidine (a substrate of horseradish peroxidase), suggesting that BSA-MnO2 NPs have enzyme-like activity. This enzyme-mimicking activity is reported elsewhere42, and demonstrate that the BSA-MnO2 NPs have been successfully prepared in this study (Figure S3A and S3B). We prepare the antibody-functionalized MnO2 nanoassembly using click reaction between Tz and TCO. The antibody functionalized-MnO2 nano-assembly can not only recognize target molecules in samples but also improve the sensitivity of this MnO2-T1 sensor through increasing the binding amount of MnO2 to a single target in the immune-reaction. The MnO2 NPs are synthesized using BSA as a template which provides lots of functional groups to react with TZ/TCO to prepare the MnO2 nanoassembly. We further utilize both mass spectrometry and DLS to characterize BSA-MnO2-TCO conjugates, BSAMnO2-Tz conjugates, antibody-Tz conjugates (Ab2-Tz), and MnO2 nano-assembly. The molecular weights (MWs) of the BSA-MnO2, BSA-MnO2-Tz, BSA-MnO2-TCO, Ab2 and Ab2-Tz are determined to be 66734 Da, 67860 Da, 74225 Da, 149220 Da, and 151440 Da, respectively. According to the MWs of TCO (514 Da) and Tz (413 Da), the coupling ratios of BSA-MnO2-Tz conjugate, BSA-MnO2TCO conjugate and Ab2-Tz are calculated to be 3:1 (Figure 3B), 15:1 (Figure 3C), and 5:1 (Figure 3E). The mass spectrometry results prove that the BSA-MnO2-TCO and BSAMnO2-Tz conjugates have been successfully prepared. The DLS study indicates that the size of MnO2 nanoassembly (about 1000 nm) is much larger than the single BSA-MnO2-Tz conjugate (about 15 nm) and BSA-MnO2TCO conjugate (about 19 nm) (Figure 3F), which further confirms the successful click reaction between Tz and TCO.

Figure 3. Characterization of BSA-MnO2-TCO conjugates, BSA-MnO2-Tz conjugates, Ab2-Tz and MnO2 nano-assembly by MS and DLS. (A)-(E) The MS spectra of BSA-MnO2 NPs, BSA-MnO2-Tz conjugates, BSA-MnO2-TCO conjugates, Ab2, Ab2-Tz conjugates, respectively; (F) DLS analysis of BSAMnO2 NPs before and after click reactions.

Sensitivity and selectivity. To obtain the best performance of the MnO2-T1 sensor, we optimize the assay conditions including the concentration of BSA-MnO2-Tz, BSA-MnO2-TCO, MBs-Ab1 conjugate, AA, and the reaction time. To prepare the MnO2 nano-assembly using TCO/Tz-mediated click reaction, it is important to control the molar ratio of BSA-MnO2-Tz to BSA-MnO2-TCO because it determines the signal amplification effect of the click reaction. The optimal concentration of BSAMnO2-Tz and BSA-MnO2-TCO is 2 µg/mL and 32 µg/mL, respectively (Figure S4A and S4B). When the amount of Ab2-Tz is 0.4 mg and the concentration of MBs-Ab1 conjugate is 0.6 mg/mL, ΔT1 has the highest signal (Figure S4C and S4D). We optimize the concentration of AA (99.05 µg/mL) and the time (8 min) of AA-mediated reduction reaction to obtain the best sensitivity (Figure S4E and S4F).

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the low concentration range). We investigate the performance of conventional T2-based MRS for PCT detection. In conventional MRS, the ΔT2 increases when the concentration of PCT is from 1 to 103 ng/mL, with a linear response of 5-103 ng/mL(Y=64.2X-21.2), and a LOD of 0.82 ng/mL (Figure 4A and 4B). The sensitivity of MnO2-T1 sensor for detection of PCT improves by 33 folds compared with conventional MRS, and the linear range of MnO2-T1 sensor is one order of magnitude broader than that of conventional MRS. Two main factors attribute to the high sensitivity of MnO2-T1 sensor: (1) Click reactions can increase the binding of MnO2 NPs to the target in the immuno-reaction. (2) The release of numerous Mn2+ from the MnO2 nano-assembly increases the coordination number of Mn with the surrounding water molecules. The large amounts of Mn2+ (with high relaxivity) can generate strong T1 signal to improve the sensitivity. It is noteworthy that the ΔT2 decreases when the concentration of PCT reach 2000 ng/mL in conventional MRS (Figure 4A), which is called “hook effect”, indicating that the excess antigen inhibits the aggregation of Ab2-MNPs conjugate and results in a low ΔT2 in conventional MRS. In contrast, the MnO2-T1 sensor overcomes this limitation of “hook effect” because the T1 signal only relates to the amount of MnO2 nano-assembly. This MnO2-T1 sensor is advantageous to conventional MRS in terms of sensitivity, linear range and accuracy. Meanwhile, the selectivity test for PCT detection shows that the ΔT1 is much higher than other proteins (Figure 4C), suggesting the good specificity because of the highly specific immuno-recognition. Real serum sample analysis. We employ MnO2-T1 sensor for PCT detection in the real serum samples. The PCT levels in serum samples are pre-determined by the electro-chemiluminescence immunoassay (ECI) with an automated analyzer (Roche-ECL, Cobas E411, Roche Diagnostics). The clinical cut-off value of PCT detection is 0.5 ng/mL. Sample 1, 2, 6, 7 and 11 are negative by both the MnO2-T1 sensor and the Roche-ECL method, and other samples are positive by both methods (Figure 5A). In addition, the measured PCT levels by the MnO2-T1 sensor show good consistency with the Roche-ECL method (Figure 5B, Table S1). However, Sample 8 to 10, 12 to 14 are negative by the conventional MRS due to the insufficient sensitivity (Figure 5A), demonstrating the better sensitivity of this approach than conventional MRS. With a better accuracy, the MnO2-T1 sensor shows great potential for detection of trace targets in clinical diagnosis.

Figure 4. The sensitivity and selectivity of MnO2-T1 sensor for detection of PCT. (A) The standard curve of MnO2-T1 sensor and conventional T2-MRS sensor for PCT detection in PBS solution (30% fetal calf serum). The concentration of PCT is from 0.01 ng/mL to 5000 ng/mL. (B) The linear range of MnO2-T1 sensor and conventional T2-MRS sensor for PCT detection. (C) The selectivity of MnO2-T1 sensor for detection of PCT. The concentration of PCT, IL-6, CRP and IgG is 500 ng/mL, 3000 ng/mL, 50 µg/mL and 50 µg/mL, respectively.

Under the above optimized conditions, we employ the MnO2-T1 sensor to detect procalcitonin (PCT), an important biomarker for bacterial infection. The concentration of PCT in serum can reflect the degree of bacterial infection because its concentration increases when the body suffers from bacterial infection43. For PCT ranging from 0.01 to 5000 ng/mL, the ΔT1 accordingly increases (Figure 4A), and a linear relationship between ΔT1 and the concentration of PCT ranges between 0.2 ng/mL and 103 ng/mL(Y=116.7X+110.6) (Figure 4B). The limit of detection (LOD) of MnO2-T1 sensor for detection of PCT is 0.025 ng/mL (LOD=3S/M, S: the value of the standard deviation of blank samples; M: the slope of standard curve within

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Figure S4. Optimization of conditions of MnO2-T1 sensor for detection of PCT

AUTHOR INFORMATION Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank the National Science Foundation of China (81671784, 21505027, 81361140345, 21535001, 81730051, 21761142006), the Ministry of Science and Technology of China (2013YQ190467), Chinese Academy of Sciences (XDA09030305, 121D11KYSB20170026).

REFERENCES Figure 5. The results of MnO2-T1 sensor, Roche-ECL method and T2-MRS sensor for detection of PCT in real serum samples. (A) The results of MnO2-T1 sensor, Roche ECL method and T2-MRS sensor for detection of PCT in real serum samples; (B) The comparison of PCT levels measured by the MnO2-T1 sensor and Roche-ECL, showing a good consistency between two methods.

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CONCLUSION We develop a T1-based nanosensor by using Mn2+induced T1 signal as readout. Mn2+ released from the reduction of MnO2 nano-assembly can dramatically induce the signal change of T1, which greatly improves the sensitivity and overcomes the problem of “hook effect” in conventional MRS. This MnO2-T1 sensor not only retains the advantage of conventional MRS but also greatly improves its sensitivity and stability, which provides an attractive magnetic sensing strategy for bioassay. In further work, we will focus on developing a multiplex analysis strategy to improve the detection efficiency of this sensor for clinical diagnosis.

ASSOCIATED CONTENT Supporting Information Supporting information include materials and equipment and additional analytical data as follows. Figure S1-S4 Table S1 Table S1: The results of MnO2-T1 sensor, Roche-ECL method and T2-MRS sensor for detection of PCT in real serum samples. Figure S1: The change of T1 value upon the addition of HCl into BSA-MnO2 solution. Figure S2: The effect of ascorbic acid (AA) to magnetic beads. Figure S3: Characterization of the enzyme-like activity of BSA-MnO2 NPs.

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For TOC use only

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T1-Mediated Nanosensor for Immunoassay Based on an Activatable

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