Metal Organic Frameworks Combining CoFe2O4 Magnetic

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Metal organic frameworks combining CoFe2O4 magnetic nanoparticles as highly efficient SERS sensing platform for ultrasensitive detection of N-terminal pro-brain natriuretic peptide Yi He, Yue Wang, Xia Yang, Shunbi Xie, Ruo Yuan, and Yaqin Chai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01112 • Publication Date (Web): 08 Mar 2016 Downloaded from http://pubs.acs.org on March 11, 2016

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ACS Applied Materials & Interfaces

Metal organic frameworks combining CoFe2O4 magnetic nanoparticles as highly efficient SERS sensing platform for ultrasensitive detection of N-terminal pro-brain natriuretic peptide Yi He, Yue Wang, Xia Yang, Shunbi Xie, Ruo Yuan∗, Yaqin Chai∗ Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University）, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China Corresponding Authors. E-mails: [email protected] (R.Yuan), [email protected] (Y. Q. Chai). Fax: +86-23-68253172. Tel.: +86-23-68252277.

ABSTRACT: N-terminal pro-brain natriuretic peptide (NT-proBNP) has been demonstrated to be a sensitive and specific biomarker for heart failure (HF). Surface-enhanced Raman spectroscopy (SERS) technology can be used to accurately detect NT-proBNP at an early stage for its advantages of high sensitivity, less wastage and time consumption. In this work, we have demonstrated a new SERS-based immunosensor for ultrasensitive analysis of NT-proBNP by using metal-organic frameworks (MOFs)@Au Tetrapods (AuTPs) immobilized toluidine blue as SERS tag. Here, MOFs@AuTPs complexes were utilized to immobilize antibody and Raman probe for their excellent characteristics of high porosity, large surface area and good biocompatibility which can obviously enhance the fixing amount of biomolecule. In order to simplify the experimental operation and improve the uniformity of the 1

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substrate,

Au

nanoparticles

functionalized

CoFe2O4 magnetic

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nanospheres

(CoFe2O4@AuNPs) were further prepared to assemble primary antibody. Through sandwiched antibody-antigen interactions, the immunosensor can produce a strong SERS signal to detect NT-proBNP fast and effectively. With such design, the proposed immunosensor can achieve a large dynamic range of 6 orders of magnitude from 1 fg mL-1 to 1 ng mL-1 with a detection limit of 0.75 fg mL-1. And this newly designed amplification strategy holds high probability for ultrasensitive immunoassay of NT-proBNP. Keywords: N-terminal pro-brain natriuretic peptide, metal-organic frameworks, CoFe2O4 nanoparticles, SERS, immunoassay 1

Introduction Currently, heart failure (HF) is a global health problem for its incidence, prevalence and high mortality rate. Now more than 23 million people are diagnosed with this illness around the world.1 Early and precise diagnosis of HF can reduce the morbidity, mortality and costs associated with hospitalisations2-5, so it is important to find an efficient predictor for diagnosis of HF. Recently, N-terminal pro-brain natriuretic peptide (NT-proBNP), a peptide released from myocardium in answer to ventricular wall stress and dysfunction, has been established as a new diagnostic and prognostic marker of HF.6 So far, many traditional immunoassay methods have been developed to detect NT-proBNP, such as electrochemical7, electrochemiluminescence (ECL)1, enzyme linked

2


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immunosorbent assay (ELISA)8 and mass spectrometry (MS)9. Although these methods possess good specificity, when detecting ultra trace concentration of NT-proBNP, they often suffer from poor sensitivity and low precision. Surface-enhanced Raman scattering (SERS), an ultrasensitive vibrational spectroscopic technique, can directly detect picomoles to femtomoles of analyte in samples.10, 11 Owing to the superiorities of less sample consumption, rapid response, noninvasive analysis, high sensitivity and specificity12, 13, SERS-based immunoassay technique has already attracted more and more attention in the field of biological analysis. The “SERS tags”, consists of metallic nanoparticles and specific organic Raman reporter molecules14, play an important role for the generation and amplification of Raman signals to achieve the purpose of targets detection. Therefore, a variety of SERS tags using different structures of nanomaterials have been developed for construction of SERS-active biosensors, such as various shapes of gold nanoparticles15-18, Au−Ag core-shell structure19, 20, nanohollows21, 22 and aggregates23. Nevertheless, these compounds are too easily aggregated to connect enough antibodies and signal molecules, which cannot meet the requirement of high sensitivity for immunosensor. Thus searching for a new type of multifunctional nanomaterial as SERS tags with high surface area, good biocompatibility is highly desired to improve the sensitivity of the biosensor.

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Metal-organic frameworks (MOFs), regularly assembled by metal ions and organic ligands linked together through a coordination interaction, are a new kind of highly porous crystalline material24, 25. Due to the advantages of their high surface area, tailorable chemistry, uniform and tunable nanostructured cavities26, MOFs are attractively potential applied in analytical chemistry26-30. MOFs can be also used as a shell of SERS-active nanomaterial to accommodate more Au or Ag nanoparticles for its high porosity, large surface area and good stability. Most representatively, Li et al. synthesized one functional material with Au nanoparticles embedded inside MOFs nanostructure achieving a dramatic enhancement of Raman intensity.31 This provides us a new field of vision to search new materials used in SERS. Herein, the amino functionalized MOF-3 (IRMOF-3), in which amino groups are exposed outside of 3D framework, was employed to prepare IRMOF-3@Au tetrapods (AuTPs)@toluidine blue (TB) as SERS tags in a SERS-based sandwich immunosensor for detection of NT-proBNP in this work. The compounds (IRMOF-3@AuTPs) were prepared through the specific covalent bond between Au and amino group, which were further used to immobilize SERS reporter (TB) and second anti-NT-proBNP (Ab2) to form IRMOF-3@AuTPs@TB@Ab2. This SERS tag can obviously improve the sensitivity of the proposed biosensor because of the large surface area of IRMOF-3 and hotspots provided by AuTPs. To simplify the experimental

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operation33 and concentrate the analytes for further improving the sensitivity of the biosensor, Au nanoparticles functionalized magnetic CoFe2O4 nanoparticle (CoFe2O4@AuNPs) was employed to immobilize primary antibodies (Ab1) forming

CoFe2O4@AuNPs@Ab1.

IRMOF-3@AuTPs@TB@Ab2

In

the

complexes

presence were

of

NT-proBNP,

coupled

with

CoFe2O4@AuNPs@Ab1 via antigen-antibody immunoreaction. For this strategy, the Raman signal of the TB increased proportionally with increasing concentration of NT-proBNP. Based on the novel magnet combining MOFs SERS sensing platform (magnet/MOFs), the proposed immunosensor showed good sensitivity with wide linear range, providing a promising potential in future trace protein immunoassay. 2

Materials and methods 2.1 Materials and regents Lysine was purchased from Ruji Biochemical Reagent Co., Ltd (Shanghai, China).

Bovine

serumal

bumin

(BSA),

gold

2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic

chloride

acid

(HAuCl4),

(HEPES)

were

obtained from Sigma Chemical Co. (St.Louis, MO). Toluidine blue (TB), Zinc nitrate hexahydrate (Zn (NO3)2·6H2O), polyvinylpyrrolidone (PVP), N, N-Dimethylformamide (DMF), 2-amino terephthalic acid (NH2-H2BDC), ferric chloride (FeCl3·6H2O), Cobaltous nitrate (Co (NO3)2·6H2O), Sodium hydroxide (NaOH), and ethylene glycol were purchased from ChengDu Kelong Chemical

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Reagent Company (Chengdu, China). And the N-terminal pro-brain natriuretic peptide (NT-proBNP) antibodies and antigens from human semen were obtained from Southwest hospital (Chongqing, China). All other chemicals were of reagent grade and used as received. The solutions used in this experiment were prepared using ultrapure water (specific resistance of 18 MΩ·cm). 2.2. Apparatus Transmission electron micrograph images were recorded on a Tecnai G2 F20 S-Twin 200 KV microscope (TEM, FEI, USA). The morphologies of different nanoparticles were tracked by a scanning electron microscope (SEM, S-4800, Hitachi). The UV-vis absorption spectra were recorded in the range of 200-700 nm using a UV-vis spectrometer (UV-vis 8500, China). The crystalline structures were characterized by X-ray diffraction (XRD, MAXima_X XRD-7000, Japan) with Cu Kα radiation at a scan speed of 5o min-1. SERS spectra were obtained using a Renishaw Invia Raman spectrometer with 50-objective. The excitation wavelength was 785 nm from a 280-mW, 785-nm laser. Raman spectrometer was calibrated by a silicon wafer at 520 cm-1 Raman shift before SERS measurement. All the spectra were the results of a single 10 s accumulation. 2.3 Preparation of IRMOF-3@AuTPs@TB@Ab2 The IRMOF-3 was prepared according to the literature34 with a little modification. Briefly, 0.20 g PVP was dissolved into the mixed solvent including 4 mL DMF and 4 mL ethanol, and then 66.93 mg Zn(NO3)2 and 16.29 mg

6


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NH2-H2BDC dissolved in 4 mL DMF were added into the above solution. Afterwards, the solution was sonicated for 20 min and then transferred into a Teflon-lined stainless steel autoclave to heat at 120 oC for 16 h. Finally, the obtained products were collected by centrifugation at 8000 rpm for 15 min and washed with DMF, ultrapure water alternately. The products were dispersed into ultrapure water for further use. The Au tetrapod was synthesized according to the references35. Firstly, 3 M aqueous HEPES solution was prepared by dissolving 9.54 g HEPES powder into 20 mL ultrapure water, and subsequently 1 M NaOH was used to adjust the pH value of the buffer solution to 7.0. Secondly, 137 µL 1% HAuCl4 was added into the above solution with stirring for 10 min continuously. The products were collected by centrifugation and then washed with ultrapure water several times. After that, 1 mL obtained Au Tetrapods were added into 2 mL IRMOF-3 solution with vigorous stirring for 2 h. Then the deposits (IRMOF-3@AuTPs) were subject to centrifugation and redispersed in 2 mL of ultrapure water for further use. TB was used as SERS reporter in this work. Firstly, 200 µL of as prepared 1 mM TB solution was added into 200 µL IRMOF-3@AuTPs solution, and reacted for 2 h under magnetic stirring. After the TB had been adsorbed onto the surface of the Au tetrapod through the interaction between Au and amino groups, the resulting solution was centrifuged at 16000 rpm for 15 min and washed with

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ultrapure water for several times to remove the unbound TB. Subsequently, the resulting particles were dispersed in 2 mL PBS (pH = 7.4) containing 1.34 µg mL-1 Ab2 for 12 h under mild magnetic stirring at 4

o

C. The

IRMOF-3@AuTPs@TB@Ab2 signal tags were obtained and collected through centrifuged at 16000 rpm for 15 min three times and finally dispersed in 2 mL PBS (pH = 7.4). AuTPs@TB@Ab2 nanocomposites were synthesized by a similar way. 2.4 Preparation of CoFe2O4@AuNPs@Ab1 The CoFe2O4 magnetic material was prepared according to literature36. Typically, 0.88 g Lysine was dissolved into 25 mL ethylene glycol under magnetic stirring, then 0.27 g FeCl3·6H2O and 0.29 g Co(NO3)2·6H2O was orderly added into Lysine solution under magnetic stirring until forming homogenous solution. 0.36 g NaOH was dissolved in another 15 mL of ethylene glycol under magnetic stirring, and subsequently the NaOH solution was dropwise added into the solution containing Lys, iron salt and cobalt salt. After continuously stirring for 20 min, the mixture was transferred to a Teflon-lined stainless steel autoclave and heated at 180 oC for 10 h and finally cooled to room temperature. The deposits were collected by centrifugation at 8000 rpm for 15 min, washed alternately with ultrapure water and ethanol, and then dispersed in ultrapure water for further use.

8


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The CoFe2O4@AuNPs nanocomposites were synthesized including three steps. Firstly, 50 µL of 0.28 mM APTMS was added into the CoFe2O4 solution under magnetically stirring for 2 h to obtain NH2-functionalized CoFe2O4. Secondly, 1 mL of 1% HAuCl4 was dropped into 2 mL of functionalized CoFe2O4 solution and the solution was stirred for 10 min. Thirdly, 1 mL of 0.1 M NaBH4 solution was slowly added under the condition of vigorous stirring, and the deposits were collected by centrifugation, then washed with ultrapure water several times. The obtained CoFe2O4@AuNPs nanocomposites were dispersed in 2 mL PBS (pH = 7.4) containing 1.34 µg mL-1 Ab1 for 12 h under mild magnetic stirring at 4 oC. Finally CoFe2O4@AuNPs@Ab1 nanocomposites were collected through magnetic separation and washed twice with PBS (pH = 7.4). 2.5 Sandwich-immunoassay protocol The immunoassays were conducted through a typical sandwich-type assay. 10 µL CoFe2O4@AuNPs@Ab1 and 10 µL BSA was dropped into a centrifuge tube mixed with 30 min. After that, the products were washed by magnetic separation three times. Subsequently, 10 µL NT-proBNP standard solutions of different concentrations was dropped into 10 µL CoFe2O4@AuNPs@Ab1 solution incubated within 40 min at room temperature. After washed with ultrapure water by magnetic separation, 10 µL IRMOF-3@AuTPs@TB@Ab2 was mixed with acquisition and incubated for 40 min at room temperature and then rinsed with ultrapure water three times. Finally, 10 µL final immune products were dropped

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on to silica wafers (1 cm×1 cm) and there was a cylindrical magnet (diameter ~ 2 mm, length ~ 5 mm) under the silica wafer to magnetically concentrate the determinants into a small spot. Subsequently, the substrates were dried under a stream of nitrogen before the SERS measurement. Scheme 1 shows the schematic illustration of the sandwich immunosensor fabrication process.

Scheme 1. Schematic diagram of the SERS-based immunosensor for the detection of NT-proBNP.

3

Results and discussion 3.1. Characterization of materials As shown in Figure 1A, the SEM image of IRMOF-3 revealed that uniform microspheres were obtained with size of 1~2 µm, which were composed of flakes. TEM image (Figure 1C) proved further evidence for sphere shape of IRMOF-3. When Au tetrapods were covalent bonded with IRMOF-3, the morphology (Figure 1B) of MOFs had a little change that the flakes cannot be 10


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seen clearly. From the magnifying image (inset of Figure 1B), some of the light spots appeared, which color was different from MOFs implied that the Au tetrapods had already assembled on MOFs. From the TEM image (Figure 1D), Au tetarpods can be obviously seen on MOFs, which further confirmed the successful preparation of IRMOFs-3@AuTPs.

Figure 1. SEM and TEM images of MOFs (A and C) and MOFs@AuTPs (B and D), Inset of Figure B: enlarged images of Figure B.

The XRD were also characterized to further prove the IRMOF-3. Figure 2 revealed that the pattern of IRMOF-3 is very similar to those reported in the literature34, 37, which can prove the successful synthesis of IRMOF-3. Besides, the N2 physisorption measurements were tested to demonstrate IRMOF-3 have the average pore size of 5.35 nm and the BET surface area of 5.89 m2/g. 11



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Figure 2. XRD of IRMOF-3.

As shown in Figure 3 (A and B), the SEM and TEM images of CoFe2O4 magnet nanoparticles revealed that the nanospheres were possessed of good uniformity with an average size of about 80 nm. The phase structure of the CoFe2O4 was characterized through powder XRD. The XRD patterns were illustrated in Figure 3C, in which all peaks can be assigned to the corresponding phased of CoFe2O4 (JCPDS card NO. 03-0864). As shown in Figure 3D, When AuNPs were in-situ reduced onto CoFe2O4, a strong absorption peak at 570 nm can be obviously found in the UV-Vis spectrum of CoFe2O4@AuNPs (Figure 3D (black)), which was different from spectrum of CoFe2O4 in Figure 3D (red). And this absorption peak is index to AuNPs which can prove that Au nanoparticles have already reduced on the CoFe2O4 nanospheres.38

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Figure 3. SEM and TEM images of CoFe2O4 (A and B), XRD of CoFe2O4 (C) and UV-visible spectra of CoFe2O4 (red) and CoFe2O4@AuNPs (black) (D).

The magnetic property of the CoFe2O4 nanoparticles was measured using a vibrating sample magnetometer at room temperature. The magnetization curves of CoFe2O4 nanoparticles were depicted in Figure 4A. Almost no hysteresis loop and zero coercivity were observed at room temperature, suggesting that CoFe2O4 nanoparticles were of the superparamagnetic nature36. Moreover,

the

photographic images of CoFe2O4 before and after adding external magnetic field were shown in Figure 4B. We can find that the prepared CoFe2O4 nanoparticles can be easily dispersed in the solution and form a stable suspension (before).

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With the external magnet, however, CoFe2O4 were aggregated together (after), indicating the prepared CoFe2O4 have magnetic response.

Figure 4. Room-temperature magnetization curves of CoFe2O4 nanoparticles (A), the photographic images of the magnetic separation using magnet (B).

3.2 Raman enhanced principle of the immunosensor To prove the feasibility of the experiment, we measured Raman peak of IRMOF-3 and their complexes. Figure 5A showed that the Raman peak of IRMOF-3 and IRMOF-3@AuTPs were very weak (black and blue). However, the peak became obvious (red) when TB was collected on IRMOF-3@AuTPs, especially the peak at 1420 cm-1 which is located from CH bending. The results indicated that TB can be used as a SERS molecular beacon in this work. The loading amount of TB was also

a

key

parameter

for

Raman

signal.

TB

was

connected

with

IRMOF-3@AuTPs via amine-Au covalent bond to form stable compounds (IRMOF-3@AuTPs@TB). In our experiments, we used excess TB to reach saturation loading amount. And the amount of TB would keep stable relatively during

the

immunoassay.

Similarly,

we

used

the

same

amount

of

14


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IRMOF-3@AuTPs in each experiment to ensure the consistency of the experiment. We also tested the SERS spectra with different batches of the same concentration of NT-proBNP after the immunoassay, the results were shown in Figure 5B. From Figure 5B, it can be easily seen that the peak of TB were basically stable at the same conditions with different batches, which indicated the amount of TB and IRMOF-3@AuTPs would not affect the effectively of the biosensor. Afterwards, CoFe2O4@AuNPs and IRMOF-3@AuTPs@TB were utilized to fix Ab1 and Ab2.

In this process, excess Ab1 and Ab2 were added to

CoFe2O4@AuNPs and IRMOF-3@AuTPs solution separately to achieve a saturation load. We also tried our best to guarantee that the experiment is basically consistent at each time. When IRMOF-3@AuTPs@TB@Ab2 was coupled with CoFe2O4@AuNPs@Ab1 through antigen-antibody immunoreaction, an obvious Raman signal is obtained. There may be two factors: One is the IRMOF-3@AuTPs which can directly enhance the TB to produce a distinct signal. The other is CoFe2O4@AuNPs which can play the role of magnet to concentrate the products and further enhance the signal of TB. Considered the two factors, the immunosensor can achieve a good sensitivity and low detection limit.

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Figure

5.

SERS

spectra

of

IRMOF-3

(black),

IRMOF-3@AuTPs

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(blue),

IRMOF-3@AuTPs@TB (red) (A), SERS spectrum measured from five different batches but the same conditions after sandwich immunoassay at 1420 cm-1: CNT-proBNP = 1 pg mL-1 (B).

3.3 Analytical performance of the SERS-based immunosensor for detection of NT-proBNP To exploit the sensitivity and potential quantitative application of the proposed immunosensor, we measured standard samples of NT-proBNP with different concentrations using the developed sandwich-type with IRMOF-3 and CoFe2O4 magnet material as SERS sensing platform. Figure 6 showed the Raman signal responses of different concentrations of NT-proBNP (A) and calibration curve of NT-proBNP (B). From Figure 6A, we could see that the Raman signal intensity gradually increased when the concentration of NT-proBNP elevated from 1 fg mL-1 to 1 ng mL-1 with a good reproducibility (each concentration for three times). Figure 6B (curve a) showed a nice linear relationship between the Raman intensity at 1420 cm-1 and the logarithm of NT-proBNP concentrations. The regression equation is y = 5456.6lgc + 24981.5 (where y is the peak intensity at 16


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1420 cm-1, c is the concentration of NT-proBNP) with a correlation coefficient square of 0.996. The detection limit for NT-proBNP calculated according to IUPAC recommendation was 0.75 fg mL-1 at 3σ

39,40

. To highlight the role of

MOFs in this proposed immunosensor, we constructed a new immunosensor for comparison. When in the absence of MOFs, which meant only Au tetrapods@TB played the role of SERS tags, AuTPs@TB@Ab2 as comparisons was constructed with sandwich immunosensor at the same way. The relationship between Raman signal responses and concentration of NT-proBNP were also evaluated as seen in Figure 6B (curve b). Figure 6B showed the peak intensity at 1420 cm-1 is increased with the increasing concentration of NT-proBNP from 1 pg mL-1 to 1 ng mL-1. And the detection limit was 0.37 pg mL-1 which was much higher than MOFs-based immunosensor. This result indicated that MOFs combining CoFe2O4 magnetic nanoparticles as SERS-active supporting substrate can enhance the immobilization amount of AuTPs and Ab1, leading a dramatic enhancement of Raman intensity and reducing the detection limit of the immunosensor finally. In addition, the analytical performance of the developed immunosensor for NT-proBNP detection has been compared with other previously biosensors reported in the literatures. From Table 1, our proposed immunosensor showed a much higher sensitivity and wider linear range, which provided a cogent evidence of our strategy for highly sensitive detection of

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NT-proBNP. Therefore, this magnet/MOFs SERS sensing platform can obviously and effectively improve the sensitivity of the immunosensor.

Figure 6. (A) the SERS-based immunosensor with the increasing concentration of NT-proBNP (pg mL-1) from a to h: (a) 0, (b) 0.001, (c) 0.01, (d) 0.1, (e) 1, (f) 10, (g) 100, (h) 1000; (B) The calibration plot of Raman intensity vs. lgc at 1420 cm-1 (error bars = SD, n = 3): (a) immunosensor used IRMOF-3@AuTPs@TB as SERS tags and (b) immunosensor used only AuTPs @TB as SERS tags. Table 1. Proposed SERS-based immunosensor performance compared with other biosensors for NT-proBNP detection.

analytical method

Detection limit

ECL ECL cyclic voltammetry microfluidic immunoassay SERS

-1

3.86 fg mL 1.67 pg mL-1 6 pg mL-1 3 pg mL-1 0.75 fg mL-1

liner range

refs -1

0.01-100 pg mL 0.005-25 ng mL-1 0.02-100 ng mL-1 0.005-4 ng mL-1 0.001-1000 pg mL-1

1 41 7 42 this work

3.4. Reproducibility of the SERS-based immunosensor SERS spectra of the MOF-based immunosensor at fifteen different spots were collected and the intensity at 1420 cm-1 was used to test the reproducibility. From 18


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Figure 7A, we can easily see that the SERS spectra were basically consistent. The coefficient of variation was less than 6% shown in Figure 7B. This result indicated that the magnet/MOFs SERS sensing platform possess good reproducibility.

Figure 7. SERS spectrum measured from fifteen different spots (A) and the intensity at 1420 cm-1 (B) (CNT-proBNP = 1 pg mL-1).

3.5. Selectivity of the SERS-based immunosensor To evaluate the selectivity of the SERS-based immunosensor, we challenged the immunosensor with other possible interferences such as human alphafetoprotein (AFP), carcinoembryonic antigen (CEA), glucose (GLU), human serum albumin (HSA) and immunoglobulin G (IgG) in the same conditions. And the results are exhibited in Figure 8. When the immunosensor was incubated with 100 pg mL-1 AFP, CEA, GLU, HAS and IgG solution, respectively, no apparent change of the peak intensity was observed compared to the blank test (no target molecular) in the same testing conditions. However, when NT-proBNP (1 pg mL-1) was coexisted with the interferences (mixture), the Raman spectrum response was

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almost the same as that with only NT-proBNP. All these results indicated that the proposed immunosensor has a good specificity for NT-proBNP.

Figure 8. Selectivity of the proposed SERS-based immunosensor. The concentrations of AFP, CEA, GLU, HAS and IgG were 100 pg mL−1. The mixture is containing AFP (100 pg mL−1), CEA (100 pg mL−1), GLU (100 pg mL-1), HSA (100 pg mL−1), IgG (100 pg mL−1) and NT-proBNP (1 pg mL−1).

3.6. Analysis of Clinical Serum Specimens. We evaluated the recovery in different concentrations of NT-proBNP solutions diluted in a healthy human real serum sample (acquired from Xinqiao Hospital of Third Military Medical University, China) three times. (Table 2) The obtained results showed satisfactory recoveries in the range of 90.66% to 105.1% and the RSD values from 0.9% to 5.4%. The results suggested that the immunosensor has a promising potential application for detection of NT-proBNP in clinical analysis.

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Table 2 Determination of NT-proBNP added in human blood serum (n = 3) with the proposed biosensor.

Serum Sample 1 2 3 4 5 4

Concentration of Concentration obtained NT-proBNP with immunosensor Recovery / % / pg mL-1 added / pg mL-1 0.01 0.009403 94.03 0.1 0.1051 105.1 1 0.9066 90.66 10 10.11 101.1 100 93.25 93.25

RSD / % 5.4 4.8 3.0 3.6 0.9

Conclusions In conclusion, a new SERS-based sandwich immunosensor was constructed for ultrasensitive detection of NT-proBNP using magnet/metal organic frameworks as highly efficient SERS sensing platform. This SERS-based immunosensor has multiple advantages including a simple analysis procedure (using magnetic CoFe2O4), small sample consumption (minimum of 10 µL) and high sensitivity (the detection limit of 0.75 fg mL-1), which resulted from these following reasons. First, SERS tags IRMOF-3@AuTPs@TB can obviously improve the sensitivity of immunosensor because of the large surface area of IRMOF-3 and hotspots produced by AuTPs. Second, magnetic compound CoFe2O4@Au can concentrate the analytes to further improve the sensitivity of immunosensor and simplify the experimental operation. Moreover, sandwiched antibody-antigen interactions can improve the specificity of the proposed immunosensor. In view of these advantages, we anticipate that this high sensitive and selective method has potential to be applied in clinical applications. 21



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ACKNOWLEDGEMENTS This project has been financially supported by the NNSF of China (51473136, 21575116), the Fundamental Research Funds for the Central Universities, China (XDJK2015C099, SWU114079) and National Science Foundation for Post-doctoral scientist of China (2015M572427).

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Table of Contents For TOC only

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Metal Organic Frameworks Combining CoFe2O4 Magnetic

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