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Dec 22, 2017 - We report a versatile analytical platform for assaying multiple analytes relying on changes in longitudinal relaxation time (T1) as a r...
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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

Versatile T1‑Based Chemical Analysis Platform Using Fe3+/Fe2+ Interconversion Yiping Chen,†,§ Binfeng Yin,†,§ Mingling Dong,†,§ Yunlei Xianyu,†,§ and Xingyu Jiang*,†,‡ †

Beijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biological Effects of Nanomaterials and Nano-safety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing, 100190, People’s Republic of China ‡ The University of Chinese Academy of Sciences, 19 A YuQuan Road, ShiJingShan District, Beijing, 100049, People’s Republic of China S Supporting Information *

ABSTRACT: We report a versatile analytical platform for assaying multiple analytes relying on changes in longitudinal relaxation time (T1) as a result of Fe3+/Fe2+ interconversion. The T1 of water protons in Fe3+ aqueous solution differs significantly from that of Fe2+, allowing for the development of a generally applicable T1-based assay since many redox reactions enable the interconversion between Fe2+ and Fe3+ that can result in the change of T1. Compared with conventional magnetic biosensors, this T1-based assay is free of magnetic nanoparticles (MNPs), and the stability of T1based assay is better than conventional magnetic sensors that suffer from nonspecific adsorption and aggregation of MNPs. This T1-based assay simultaneously enables “one-step mixing” assays (such as saliva sugar) and “multiple-washing” immunoassays with good stability and sensitivity, offering a promising platform for convenient, stable, and versatile biomedical analysis

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ion, and Fe2+ has a higher T1 value than that of Fe3+ at the same concentration in an aqueous solution.31 The electron spin32,33 and long electronic relaxation time31 are two main reasons that Fe3+ is more efficient at relaxing surrounding water protons than Fe2+ (Figure 1A). The electronic structure of high-spin Fe3+ is perfectly half-filled d-orbitals, so Fe3+ has a well-isolated electronic ground state and a long electron spin relaxation (ESR), which is approximately 30 times longer in the Fe3+ than that of Fe2+.31 The significant difference in the T1 value offers an excellent platform to develop an MNPs-free magnetic analytical method using Fe3+/Fe2+ interconversion given that transformation between Fe3+ and Fe2+ is readily achieved in different types of redox reactions. Other types of paramagnetic ions have been also used as magnetic probes in magnetic resonance imaging (MRI).34−36 Gd(III), a widely used MRI probe,37,38 only has one stable oxidation state in aqueous media that is not suitable for this T1-based assay. Manganese has a number of oxidation states depending on the ligand field,39 and the T1 relaxivity can be enhanced 3-fold when Mn(III) converts into Mn(II).36 However, this amplification effect is still far from the requirement for developing sensors with high sensitivity. The Fe3+/Fe2+ interconversion may have distinct advantages compared with other paramagnetic ions in the T1-based assay. In this work, we show that the T1 values of water protons between Fe2+ and Fe3+ in aqueous solution differ significantly under the same conditions (such as concentration, pH,

lthough magnetic analytical methods based on magnetic nanoparticles (MNPs) have drawn considerable interest both in research and clinics due to their simple pretreatment of samples, allowing rapid analysis with high sensitivity,1−6 they still face some challenges. In conventional MNPs-based assays, MNPs act as the carrier of magnetic separation,7−9 magnetic/ optical probe,10,11 enzyme mimic,12,13 or signal amplifier,14,15 enabling convenient signal readout and high sensitivity of magnetic analytical methods. However, these magnetic analytical methods still suffer from two limitations: (1) The nonspecific adsorption seriously affects the accuracy of assay because of the large specific surface area of MNPs.16−18 Although it can be alleviated by various surface modification strategies,19−21 they might be not only complex and timeconsuming but also compromise the properties of MNPs. (2) The uncontrolled aggregation of MNPs22−24 also affects the accuracy of magnetic analytical methods. For example, the magnetic relaxation switching (MRS) assays based on targetinduced aggregation (or disaggregation) of MNPs, which have been successfully used to detect DNA25/microRNA, 26 viruses,27 pathogens,28 cancer cells,29 and so forth, still suffer from the aggregation of MNPs, especially in complicated biological samples, resulting in the false positive signal.24,30 Development of an MNPs-free magnetic assay can circumvent the limitations of traditional approaches in terms of nonspecific adsorption and aggregation. The longitudinal relaxation time (T1) of paramagnetic ions provides a possible solution to meet the challenges. Paramagnetic ions such as Fe3+ and Fe2+ differs in T1 of water protons in their aqueous solution. The Fe3+/Fe2+ in aqueous solution can form an aqua © XXXX American Chemical Society

Received: September 27, 2017 Accepted: December 11, 2017

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DOI: 10.1021/acs.analchem.7b03961 Anal. Chem. XXXX, XXX, XXX−XXX

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the mixture solution for 1 h at room temperature. After that, we transfer this mixture solution to a clean centrifugal ultrafiltration unit (10 kDa filter) and centrifuge at 9000 rpm for 10 min at 4 °C to remove excess biotin and ions. After three washing steps using PBS solution (pH = 7.4, 0.01 M), we collect the biotin−Ab2 conjugate and dilute it using PBS and store this conjugate at −20 °C. Preparation of MNPs−Antibody (MNPs−Ab) Conjugates. We suspend 1 mg of MNPs (30 nm in diameter) into 100 μL of activated buffer (80 nM MES, pH = 6.0). We transfer 10 μL of EDC (10 mg mL−1) and 10 μL of NHS (10 mg mL−1) into to the MNPs solution. After activation for about 30 min, we add 1000 μL of coupling buffer PBS (pH = 7.4, 0.01 M) into the activated MNPs solution, and then we divide the above mixture solution equally to two parts. We add 0.05 mg of capture antibody (Ab1) or detection antibody (Ab2) into the activated MNPs. We gently stir the mixture solution for 2 h at room temperature, and add 200 μL of 3% BSA solution for 0.5 h. We collect the MNPs−Ab conjugate from the free Ab by a SuperMag separator at 4 °C for 24 h, and then resuspend this conjugate using 1000 μL of PBST, and we also separate the MNPs−Ab in magnetic field. Finally, we resuspend the MNPs− Ab conjugate using PBS solution (pH = 7.4, 0.01 M, 0.01% BSA) and store the conjugate at 4 °C for further use. Procedure of T1-Based Assay for Detection of Glucose. We add 100 μL of glucose oxidase (GOD) (0.5 μg/mL) into the 100 μL of different concentrations of glucose (0, 0.006, 0.012, 0.024, 0.048, 0.096, 0.195, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 mM), respectively. The mixture solution incubates at 37 °C for 1 h. After that, we add 100 μL of mixed solution into 100 μL of FeCl2 aqueous solution with 0.2% BSA (4 mM) at 37 °C for 30 min. Finally, we measure the T1 value of 20 μL of the resulting mixture via the NMR analyzer, and we assay three independently prepared samples to obtain the average T1 value. We use inversion− recovery pulse sequences for T1 measurements with the following parameters: the NMR frequency, 62.16 MHz; pulse separation, 10 ms; 90° pulse width, 32 μs; 180° 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 T1 was calculated using the following equation:

Figure 1. Mechanism of the T1-based assay for both “one-step mixing” assays and “multiple-washing” immunoassays. (A) The proposed mechanism of difference in the T1 value between Fe2+ and Fe3+ in aqueous solution. (B) The difference of the T1 value between Fe3+ and Fe2+ at a series of concentrations in aqueous solution. (C) Indirect detection of biomarkers using the T1-based assay based on the interconversion between Fe2+ and Fe3+ by redox reactions for both “one-step mixing” analysis and “multiple-washing” immunoassays.

temperature, dissolved oxygen, and sample matrix)(Figure 1B). This difference in T1 value can serve as a magnetic signal to develop assays by the interconversion between Fe3+ and Fe2+ without the requirement of MNPs, providing an MNP-free T1 magnetic assay. Many biochemical markers, such as vitamin C (Vc), blood sugar, and hydrogen peroxide (H2O2), can convert Fe2+ to/from Fe3+ and result in the change of T1 (ΔT1) due to their oxidizability or reducibility (Figure 1C). A number of enzymes, such as alkaline phosphatase (ALP), an important biomarker in clinical diagnosis, can catalyze enzymatic reactions to generate products that interconvert Fe3+ and Fe2+ (Figure 1C). As enzymes such as ALP can serve as a labeling enzyme in immunoassays, this T1-based assay also allows for immunoassays of antigens/antibodies. Thus, this T1-based assay provides a new multifunctional magnetic analytical platform for many types of assays without resorting to MNPs (Figure 1C).

ΔT1 = T1blank − T1sample

where T1sample and T1blank are the average T1 relaxation times of the triplicates of the sample and blank groups (the [glucose] = 0), respectively. To assay the [glucose] in saliva and urine samples, we first diluted these samples 3-fold using PBS solution. To obtain the limit of detection (LOD), we employ the following formula:40 LOD = 3S/M (where S is the value of the standard deviation of blank samples and M is the slope of standard curve within the low-concentration range). Procedure of T1-Based Assays for Detection of ALP. We add 100 μL of different concentrations of ALP aqueous solution with 0.2% BSA (ranging from 0.2 to 500 U/L) into a 96 well plate; respectively, we add 100 μL of ascorbic acid− phosphate (20 mM) into the above ALP solution, and this solution mixture incubates at 37 °C for 1 h. We add 100 μL of FeCl3 (4 mM) aqueous solution into the above mixture solution for 30 min at 37 °C. Finally, we measure the T1 value of the resulting mixture solution using the NMR analyzer, and

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EXPERIMENTAL SECTION Comparison of T1 Value between Fe2+ and Fe3+. We use the nuclear magnetic resonance (NMR) analyzer to measure the T1 value of FeCl2 and FeCl3 aqueous solution, and the concentrations of FeCl2 and FeCl3 change from the 2.6 × 10−6 to 4.0 × 10−2 M. We obtain the average T1 value from three independently prepared samples (n = 3). Synthesis of Biotinylated Anti-AFP Antibody (Ab2). We prepare the biotin−Ab2 conjugate as follows. We dilute the biotin using 100 μL of DMF (1 mg/mL); meanwhile, we dilute the Ab2 using PBS solution (pH = 8.0, 0.01 M), and the final concentration of Ab2 is 1 mg/mL. We mix the biotin and Ab2 at a molar ratio of 20:1 (biotin to Ab2), and we also shake up B

DOI: 10.1021/acs.analchem.7b03961 Anal. Chem. XXXX, XXX, XXX−XXX

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Friendship Hospital (China) in accordance with the rules of the local ethical committee (2017-P2-099-01).

we obtain the average T1 value from three independently prepared samples (n = 3). To detect ALP in whole-blood sample and pretreated serum sample, we equally divided the whole-blood samples into two parts. We dilute one part samples 10-fold using PBS solution, and directly measure the T1 value of one by this T1-based assay. We further centrifuge the other part to prepare serum samples for analysis. We compared the T1 values from the native/ pretreated whole-blood samples. Procedure of ALP-Mediated T1-Based Assay for Immunoassay. We dilute the capture antibody (Ab1) using a carbonate buffer (0.2 M sodium carbonate/bicarbonate, pH 9.6), and the finial concentration of Ab1 is 5 μg/mL. We add this 100 μL of diluted Ab1 solution onto the bottom of the 96 well microplate (Corning). The plate incubates at 4 °C for 12 h. After three washing steps with PBST washing buffer (0.01 M PBS, with 0.5% Tween-20), we add 100 μL of blocking buffer (3% BSA in PBS) onto each well for 2 h. After three washing steps, we add 100 μL of different concentrations of α fetoprotein (AFP) (0, 0.8, 4, 20, 100, 250, 500, 1000, 2000, 5000, and 10 000 ng/mL) onto each well, and the plate incubates at 37 °C for 1 h. After three washing steps, we add 100 μL of biotinylated AFP-detection antibody (Ab2) conjugate (2 μg/mL) onto each well and we put the whole well at 37 °C for 1 h. Followed by washing three times, we add 100 μL of SA−ALP conjugate (1 μg/mL) onto the plate and set the plate at 37 °C for 0.5 h. After washing steps for three times, we add 100 μL of ascorbic acid−phosphate (20 mM) into each well, and the mixture solution incubates at 37 °C for 1 h. After that, we add 100 μL of FeCl3 (4 mM) into the above solution, and we put this mixture solution at 37 °C for 0.5 h and measure the T1 value of 20 μL of the resulting mixture solution via the NMR analyzer, and we obtain the average T1 value from three independently prepared samples (n = 3). For each interval, the ΔT1 was calculated using the following equation:

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RESULTS AND DISCUSSION This T1-based assay is a versatile analytical platform, since the interconversion between Fe2+ and Fe3+ can originate from redox reactions and enzyme-catalyzed reactions. It enables multiple types of assays including “one-step mixing” assays (such as those for blood sugar, ALP, and H2O2) and “multiplewashing” immunoassay with good stability and sensitivity. Considering that the relaxivity of Fe2+/Fe3+ changes according to the different ligands it binds with, the media of Fe2+/Fe3+ is important for the measurement of the T1 signal. We employ PBS solution with 0.2% BSA to dilute the human samples to eliminate the effect of biological media on the T1 signal. T1-Based Assay for Detection of Glucose. We employ this T1-based assay for detection of glucose in saliva samples. In GOD-catalyzed reactions, glucose is the substrate and H2O2 is the product (Figure 2A). H2O2 has strong oxidizing property

Figure 2. T1-based assays for detection of glucose. (A) The scheme of the T1-based assay for detection of glucose. (B) The response of the T1 signal to the [H2O2]. (C) The T1-based assay for detection of glucose in serum samples, urine samples, and saliva samples; the [glucose] is from 0.1 to 100 mM.

ΔT1 = T1sample − T1blank

where T1sample and T1blank are the average T1 relaxation times of the triplicates of the sample and blank groups (the [AFP] = 0), respectively. Process of T2-Mediated MRS for Detection of AFP. We transfer 50 μL of MNP−Ab1 solution (1 μg/mL), 50 μL of MNP−Ab2 solution (1 μg/mL), and 100 μL of different concentrations of AFP (0, 0.8, 4, 20, 100, 250, 500, 1000, 2000, 5000, and 10 000 ng/mL) onto the bottom of the 96 well microplate. Each mixture solution is gently shaken for 30 min. Then, we take out 20 μL of the above mixture solution and measure the T2 value by the NMR analyzer, and we obtain the average T2 value from three independently prepared samples (n = 3). For each interval, the change in T2 (ΔT2) was calculated using the following equation:

that can convert Fe2+ into Fe3+ in redox reactions, resulting in the increase of ΔT1 that directly relates to the [H2O2] (Figure 2B). In clinical diagnosis, accurate and repeatable detection of [glucose] is critical for diabetes. The saliva is easier to collect than blood and does less harm to the diabetic patients.41 However, the [glucose] in saliva is about 0.1 mM, which is beyond the sensitivity of most commercialized glucose meters for detection of glucose. T1-based assay can detect 0.1 mM of glucose in spiked saliva, while the lowest detectable concentration of the glucose meter is 1 mM (Table S1). We also successfully detect the glucose in saliva samples, urine samples, and serum samples using the T1-based assay with the limit of detection (LOD) of 0.12, 0.17, and 0.14 mM, respectively (Figure 2C). We define the LOD in this work as follows: LOD = 3S/M, where S is the value of the standard deviation of blank samples and M is the slope of standard curve within the low-concentration range. T1-Based Assay for Detection of ALP. This T1-based assay allows for detection of ALP, whose abnormal level in serum is usually associated with several diseases such as liver dysfunction, breast and prostate cancer, diabetes, and so on.42,43 ALP can catalyze dephosphorylation reactions to remove a

ΔT2 = T2blank − T2sample

where T2sample and T2blank are the average T2 relaxation times of the triplicates of the sample and blank groups (the [AFP] = 0), respectively. Human Sample Analysis. We collect the ALP human samples (whole-blood samples and serum samples) and AFP human samples (serum samples) which are from the people who may suffer primary biliary cirrhosis (PBC) from the Beijing C

DOI: 10.1021/acs.analchem.7b03961 Anal. Chem. XXXX, XXX, XXX−XXX

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detection of ALP in hemolysis-contaminated serum samples (Table S2). The T1-based assay can directly detect ALP in all these samples without complex sample pretreatment, while the [ALP] in sample 4 cannot be detected by the conventional pNPP-based method because of hemolysis in the serum samples. In contrast, this T1-based assay is suitable for analysis of turbid samples, including biological, environmental, or food samples because the T1-based assay is based on a magnetic readout and nearly no samples have any magnetic interference on the T1 signal. We detect ALP in the whole-blood samples to demonstrate the anti-interference property of this T1-based assay. The whole blood presents challenges for assays because blood cells such as erythrocytes severely interfere with both biochemical reaction and readout. Using the T1-based assay, the [ALP] in native whole-blood samples and pretreated whole-blood samples has a good coincidence with a correlation coefficient of 0.95 (Figure 3F). It suggests that the T1-based assay can directly detect ALP in whole-blood samples with an acceptable accuracy without complex sample pretreatment steps. In addition, the T1-based assay needs only 20 μL of sample, which is suitable for detection of biomarkers in microsamples. However, this T1based approach would only work for a limited set of analytes in the “one-step mixing” assay because many biological analytes do not produce redox-active products which impair the applicability of the method. In addition, the oxidizing/reducing substances in complicated samples might affect this T1-based assay given that the sensing mechanism relies on the redox reaction-based Fe3+/Fe2+ interconversion. T1-Based Assay for Detection of AFP. Besides the “onestep mixing” assays for small molecules and enzymes, this T1based assay can also specifically detect antigen/antibody in “multiple-washing” immunoassays because ALP is one of the most broadly used labeling enzymes in immunoassays (Figure 4A). To demonstrate the utility of this T1-based assay in “multiple-washing” immunoassays, we chose the AFP as a target of interest, which is a glycoprotein that serves as an important biomarker of many types of cancers in clinical diagnosis. The ΔT1 increases when [AFP] increases from 0.1 to 104 ng/mL (Figure 4B). The linear range is from 4 to 2000 ng/ mL of AFP, and the linear equation is Y = 14.2X − 4.2 (X = lg[AFP(ng/mL)], R2 = 0.99) (Figure 4B). The LOD [LOD = 3S/M = 3(0.11/0.67)] is 0.49 ng/mL, 6 times higher in sensitivity than that of the conventional ALP-based enzymelinked immunosorbent assay (ELISA) using the same antibodies (Figure 4C). In addition, the T1-based assay has good stability because both Fe2+ and Fe3+ aqueous solutions are stable at room temperature for 5 months (Figure 5A); then, the shelf life of the chemicals used in this T1-based assay is much longer than that of the enzyme substance in conventional ELISA, since the latter needs to be carefully preserved (typically needs refrigeration and avoidance of light) that is not suitable for on-site use. Thus, Fe2+ and Fe3+ aqueous solutions are more stable and less expensive, making the operation and robustness of this T1-based assay better than those of conventional ELISA. We use the coefficient of variation (CV) to evaluate the stability of the T1-based assay for detection of AFP, and CV = (standard deviation/mean) × 100%. The intrabatch CV at different concentrations of AFP is under 9.6%, and the interbatch CV is under 13.5% at different [AFP] (Table S3), which reveal the good stability of the T1-based assay for “multiple-washing” immunoassays.

phosphate group from the phosphate of ascorbic acid (2phospho-L-ascorbic, which is nonreducing), to yield the ascorbic acid (vitamin C, which is reducing) that can be used as a reducing agent. Once Fe3+ is converted into Fe2+ by Vc, T1 would increase because [Fe2+] increases, and the ΔT1 directly depends on the [ALP] (Figure 3A). The ΔT1 value increases

Figure 3. T1-based assay for detection of ALP. (A) The scheme of the T1-based assay for detection of ALP. (B) The standard curve of the T1based assay for detecting ALP; the [ALP] range is from 0.5 to 3000 U/ L; the ALP aqueous solution is with 0.2% BSA. (C) The linear range of the T1-based assay for detecting ALP is from 5 to 500 U/L. (D) Correlation of the T1-based assay and conventional pNPP-based optical method for detection of ALP in human serum samples. (E) Correlation between the detected [ALP] in whole-blood samples and serum samples by the conventional pNPP-based optical method (n = 3). (F) Correlation between the detected [ALP] in whole-blood samples and serum samples by this T1-based assay (n = 3).

when the [ALP] increases from 0.5 to 3000 U/L (Figure 3B). A linear relationship between the ΔT1 value and the [ALP] falls in the range between 5 and 750 U/L, and the linear equation is Y = 0.17X + 9.73 (X = CALP, R2 = 0.97) (Figure 3C). The LOD [LOD = 3S/M = 3(0.2/1.1)] of the T1-based assay is 0.54 U/L, which is enhanced by 5-fold in sensitivity compared with that of the conventional pNPP-based method (2.55 U/L), and the linear range of the T1-based assay for detection of ALP (5−750 U/L) is better than that of the conventional p-nitrophenol phosphate (pNPP)-based method (5−500 U/L) (Figure S1). For proof of application, we use this T1-based assay to detect ALP in 25 human serum samples that were detected by the traditional pNPP-based method in the clinical laboratory of a local hospital (Beijing Friendship Hospital). The T1-based assay has a good coincidence with the conventional pNPP-based method (Figure 3D). The conventional pNPP-based method relies on the optical signals of the colored product, which is not applicable to the red-colored whole-blood samples (Figure 3E). In some cases, the serum samples also have hemoglobin, which may affect the accuracy of assay. We study the accuracy of the conventional pNPP-based method and T1-based assay for D

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prepare the conjugates of MNPs and antibody, which acts as a magnetic probe. When targets exist, the dispersed magnetic probe could become aggregated because of the antibody− antigen recognition, which results in the change of T2 value (ΔT2). The ΔT2 relies on the concentration of targets in samples. The LOD [LOD = 3S/M = 3(0.58/0.78)] of this assay for detection of AFP is 2.2 ng/mL(Figure 4D). The sensitivity of this T1-based assay (LOD = 0.49 ng/mL) is better than that of conventional T2-mediated MRS when using the same antibodies because the ALP-mediated catalytic reaction can greatly amplify the signal. In addition, our assay can successfully avoid the hook effect of traditional T2-based MRS, namely, falsely low signal at high concentrations. In the MRS, even though at low concentrations the T2 gradually increases with the increase of concentration of analyte, the T2 decreases when the [AFP] reaches 104 ng/mL, a text-book case of the hook effect,44,45 which seriously plagues the quantitative detection of traditional T2-mediated MRS (Figure 4D). The hook effect most likely happens when the excess antigen prevents the aggregation of antibody−MNPs conjugate, which results in low T2. By contrast, the hook effect can be avoided in this T1-based assay because the T1 signal only relates to the degree of interconversion between Fe3+ and Fe2+. In addition, the MNPs−antibody probe only retains stability at 4 °C for 3 months (Figure 5B), and it may also aggregate in complex samples due to nonspecific adsorption, which affects the accuracy of T2-mediated MRS. In contrast, the Fe3+ aqueous solution can retain its stability at room temperature for five months (Figure 5A), which suggest that the robustness and stability of this T1-based assay are better than those of T2mediated MRS. We employ the T1-based assay, T2-MRS sensor, and chemiluminescence microparticle immunoassay (CMIA, a commercial kit from Abbott Laboratories) to detect AFP in the human serum samples to demonstrate the real-world application of our approach. CMIA often serves as the gold standard for immunoassay due to its high sensitivity and accuracy. The correlation coefficient between the T1-based assay and CMIA for detection of AFP in these serum samples is 0.95 (Figure 4E, Figure S2), while the correlation coefficient between the T2-mediated MRS and CMIA is 0.88 (Figure 4F, Figure S2), which shows that the accuracy of the T1-based assay for real sample analysis is better than that of the T2-MRS sensor. This result further proves that the MNPs-free strategy is an effective tool to improve the analytical performance of the magnetic analytical platform. Table S4 summarizes the sensitivity and analytical sample types of the T1-based assay and other methods for detection of different targets. T1-Based Assay Realizes the “One-Step Mixing” Assay and “Multiple-Washing” Immunoassay Simultaneously. We also integrate the “one-step mixing” assay and “multiplewashing” immunoassay for simultaneous detection of two different biomarkers. It is important and necessary to simultaneously realize “one-step mixing” assay and “multiplewashing” immunoassay in clinical diagnosis when it comes to certain diseases. Compared to the conventional “one-step mixing” assay and “multiple-washing” immunoassay assay, the T1-based assay can easily integrate “one-step mixing” assay and “multiple-washing” immunoassay in one approach only using Fe2+ or Fe3+ solution. We use this T1-based assay to detect the [ALP] and [antimitochondrial antibody] (AMA) in serum samples of individuals who might suffer from PBC. In general,

Figure 4. (A) Scheme of ALP-mediated T1-based assay for the detection of AFP. (B) The ALP-mediated T1-based assay for detection of AFP in PBS solution with 0.2% BSA. The [AFP] is from 0 to 104 ng/mL. (C) The conventional ELISA for detection of AFP in PBS solution spiked with 0.2% BSA. The [AFP] is from 0.1 to 104 ng/mL. (D) The T2-mediated MRS sensor for detection of AFP in PBS solution spiked with 0.2% BSA. The concentration of AFP is from 0.1 to 104 ng/mL. (E) Correlation of the T1-based assay and chemiluminescence microparticle immunoassay (CMIA) for detection of AFP in human serum samples. (F) Correlation of the T2-mediated MRS sensor and CMIA for detection of AFP in human serum samples. The AFP levels in serum samples are predetermined by the CMIA method in a local hospital.

Figure 5. Stability of the Fe3+ aqueous solution and MNPs−antibody probe. (A)The stability of the Fe3+ aqueous solution (1.6 mM) at room temperature and (B) MNPs−antibody probe (0.5 μg/mL) at 4 °C (* represents significant difference, t test, P < 0.05).

We compare this T1-based assay with traditional T2-based MRS in terms of sensitivity and robustness. In T2-based MRS, antibodies should be conjugated to the surface of MNPs to E

DOI: 10.1021/acs.analchem.7b03961 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry high concentrations of both ALP and AMA indicate the PBCpositive samples, while low concentrations of both ALP and AMA indicate the PBC-negative samples. However, individuals may suffer from PBC if either ALP or AMA is elevated;, thus, the simultaneous detection of both biomarkers is of great importance and can improve the accuracy of diagnosis. Samples 1−5, and samples 7−9 are both AMA-negative and ALPnegative with this T1-based assay (Table 1), in accordance with

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

ALP (U/L)

*E-mail: [email protected]. ORCID

Xingyu Jiang: 0000-0002-5008-4703

50.21 55.90 35.70 39.23 151 368.45 36.88 46.09 147.07 173.94 197.86 310.41 240.21 359.43 296.09

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.96 2.06 4.81 3.85 8.08 5.33 3.27 4.89 4.56 2.94 3.91 5.12 5.13 8.01 8.81

N N N N N P N N N P P P P P P

3.25 3.2 2.85 3.03 5.99 113.72 3.40 3.30 4.67 3.27 2.79 32.05 13.83 48.08 26.13

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.08 0.04 0.05 0.27 5.1 0.11 0.04 0.13 0.07 0.05 2.83 1.44 4.31 2.43

Author Contributions §

Y.C., B.Y., M.D., and Y.X. contributed equally to this work.

clinical outcomes

AMA (U/mL) N N N N N P N N N N N P P P P

Notes

The authors declare no competing financial interest.

N N N N N P N N N N P P P P P

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ACKNOWLEDGMENTS We thank the National Science Foundation of China (81671784, 21505027, 81361140345, 51373043, and 21535001) and Chinese Academy of Sciences (XDA09030305) for financial support.

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the clinical outcomes showing that they are PBC-negative. Sample 6, and samples 12−15 are both AMA-positive and ALPpositive, in accordance with the clinical outcomes showing that they are PBC-positive. However, both samples 10 and 11 are detected to be ALP-positive and AMA-negative, but clinically, the individual of sample 10 is PBC-negative while that of sample 11 is PBC-positive. In this case, the patient needs further examination, suggesting that the simultaneous detection of different biomarkers can provide more useful information on the patient which helps the doctor to make a decision and improve the detection accuracy clinically.

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CONCLUSION In conclusion, we developed a versatile and MNPs-free T1based assay for detection of a range of analytes based on the interconversion of Fe3+ and Fe2+ and thus the change of T1 as the signal readout. This T1-based assay not only retains the advantages of magnetic analytical assay but also simplifies the analysis with enhanced sensitivity and stability, which greatly improves the detection efficiency and reduces the cost in clinical diagnosis. However, this analytical platform currently is incapable of simultaneous determination of multiple targets in the same sample. In future work, we will focus on combining the T1-based assay with microfluidic technology to realize automatic and multiplex analysis.

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AUTHOR INFORMATION

Corresponding Author

Table 1. T1-Based Assay for Simultaneous Detection of ALP and AMA in Serum Samples sample no.

Materials and equipment and analytical data for the new compounds shown in Figures S1 and S2 and Tables S1− S4 (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03961. F

DOI: 10.1021/acs.analchem.7b03961 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.7b03961 Anal. Chem. XXXX, XXX, XXX−XXX