Metal-Organic Framework Encapsulation for Biospecimen Preservation

4Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO, 63110, USA. 5The Center for Clinical Pharmaco...
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Cite This: Chem. Mater. 2018, 30, 1291−1300

Metal−Organic Framework Encapsulation for Biospecimen Preservation Congzhou Wang,† Hongcheng Sun,† Jingyi Luan,† Qisheng Jiang,† Sirimuvva Tadepalli,† Jeremiah J. Morrissey,‡,§ Evan D. Kharasch,‡,§,⊥,¶ and Srikanth Singamaneni*,†,§ †

Department of Mechanical Engineering and Materials Science, Institute of Materials Science and Engineering, Washington University in St. Louis, Saint Louis, Missouri 63130, United States ‡ Department of Anesthesiology, §Siteman Cancer Center, and ⊥Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, Saint Louis, Missouri 63110, United States ¶ The Center for Clinical Pharmacology, St. Louis College of Pharmacy and Washington University School of Medicine, Saint Louis, Missouri 63110, United States S Supporting Information *

ABSTRACT: Handling, transport, and storage of biospecimens such as blood and urine without refrigeration are extremely challenging. This formidable challenge leads to an inevitable reliance on a “cold chain” for shipping, handling, and storage of biospecimens throughout the world. The cold chain requirement precludes biospecimen procurement from underserved populations and resource-limited settings where refrigeration and electricity are not reliable or even available. Here, we introduce a universal biospecimen preservation approach based on nanoporous material encapsulation for preserving protein biomarkers in biofluids under normal (nonrefrigerated) storage conditions. Using urinary NGAL and serum/ plasma CA-125 as the model protein biomarkers, we demonstrate that zeolitic imidazolate framework-8 (ZIF-8), a nanoporous material, encapsulation can preserve protein biomarkers in urine, serum, plasma, and blood at room temperature and 40 °C, with comparable preservation efficacy to the refrigeration method (freezing liquid samples at −20 °C). The protein biomarkers in the relevant biofluids are first encapsulated within the nanoporous crystals (i.e., ZIF-8) and then dried on paper substrates via a dry spot sample collection method, which would greatly improve biospecimen collection and handling capability in resource-limited settings. Overall, this energy-efficient and environmentally friendly approach will not only alleviate huge financial and environmental burden associated with “cold chain” facilities, but also extend biomedical research benefits to underserved populations by acquiring reliable clinical samples from regions/populations currently inaccessible.



INTRODUCTION

if not impossible, to expect patients scattered in rural areas to travel and access an extremely limited number of hospitals and clinical laboratories for screening and diagnosis.11 A feasible solution to the aforementioned challenges is a refrigeration-free biospecimen preservation technology that can be implemented in resource-limited settings (i.e., absence of electricity, refrigeration, trained personnel) and underserved populations to efficiently collect, process, and ship well-preserved biospecimens to centralized clinical/research laboratories and hospitals with qualified facilities for analysis. Considering that the Clinical and Laboratory Standards Institute (CLSI) recommends urinalysis and culture and testing within 2 h of urine sample collection, urinary samples are often treated with preservatives such as boric acid, tartaric acid, and chlorhexidine to extend the sample storage time if analysis

The availability of high-quality biospecimens such as blood and urine is critical to biomedical research and clinical diagnosis.1−3 Unfortunately, because of the poor stability of molecular biomarkers (especially proteins) in these biofluids at ambient and elevated temperatures, they are prone to lose their structure and biofunctionality during the preanalytical stage (i.e., the preparation, storage or transportation period between sample collection and analysis).4−6 For instance, when refrigeration is not maintained, blood-derived biospecimens degrade quickly, accounting for up to 67% of all laboratory testing errors.5,7,8 This requires investigators and clinicians to dedicate a significant fraction of budget to refrigeration or “cold chain” costs associated with biospecimen storage or transportation.9,10 More importantly, the refrigeration and “cold chain” are simply not feasible in resource-limited settings (e.g., low and middle income countries), which hinders biospecimen procurement, disease screening, early diagnosis, and therapeutic intervention in underserved populations since it is impractical, © 2018 American Chemical Society

Received: November 9, 2017 Revised: January 29, 2018 Published: February 1, 2018 1291

DOI: 10.1021/acs.chemmater.7b04713 Chem. Mater. 2018, 30, 1291−1300

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Figure 1. Schematic illustration depicting the concept of using MOF encapsulation for biospecimen preservation. By combining MOF encapsulation and dry spot sample collection, protein biomarkers can be preserved on a paper substrate under nonrefrigerated conditions. Before bioanalysis, the MOF-encapsulated proteins can be recovered without losing structure and function.

within 2 h of sample collection is not feasible.12,13 However, these preservatives are applicable only for short preservation times ranging from 24 to 72 h, which are not sufficient for implementation in resource-limited settings where the time lapse between sample collection and analysis can be much longer. Moreover, the preservation time scales with the existing preservatives are also not sufficient for research biospecimens used for biomarker discovery and validation, which often involves months to years. Other approaches to dry blood samples, such as isothermal vitrification,14 lyophilization,15 or within a solid matrix such as silk fibroin, have been reported.16 In the isothermal vitrification and lyophilization approaches, sugars such as sucrose and trehalose are often added into protein solution to form a sugar-glass matrix for stabilizing proteins.17 However, vitrification and lyophilization of protein with sugars are difficult to implement in resource-limited settings and in some cases lack of control of sugar crystallization can lead to protein aggregation.18 Drying blood in a silk fibroin matrix represents a novel approach for longterm biofluid preservation without refrigeration. However, the technology involves the use of silk fibroin solution with limited temperature and environmental stability and relatively short shelf life, making it difficult to implement in resource-limited settings. It is also reported that the addition of zinc ions (such as zinc sulfate) can enhance the stability of specific types of proteins.19,20 Overall, none of the existing methods can be considered a “universal biospecimen preservation technique”, which would allow both long-term storage without refrigeration and the implementation in resource-limited settings. Metal−organic frameworks (MOFs) are an emerging class of nanoporous materials that are composed of metal ions or clusters linked by organic ligands.21,22 MOFs are considered to be highly attractive for a variety of applications including, gas and energy storage,23,24 drug delivery,25−27 catalysis,28,29 separation,30,31 and chemical sensors.32 Their attractive properties include nanoporous structure with a large surface area, tunable porosity, rich organic functionality, stable shelf life of precursor materials, and excellent thermal stability.33−36 Within the emerging applications, of particular interest is the biopreservation ability of the MOFs, which is believed to rival conventional porous solids and biomaterials.37,38 When incorporated into these nanoporous materials to form MOF

biocomposites, the protein molecules will be confined within the rigid framework structures, thus maintaining their structures and activities against denaturation and degradation conditions.39,40 Pioneering work by Falcaro and Tsung’s groups involved spontaneous biomineralization process or a de novo approach to encapsulate enzymes within MOFs for biocatalysis applications.37,41,42 We and others recently demonstrated that MOF (zeolitic imidazolate framework-8, ZIF-8) can be used as a protective material to preserve the recognition capability of antibodies or viral protein on biosensor surfaces stored at ambient and elevated temperatures.43−45 The preserved antibody-based biosensor can be restored at a later time (before use for detection) by dissociating the ZIF-8 layer in an aqueous solution at pH 6. Considering the versatile molecular encapsulation capability of ZIF-8 in aqueous solution, high thermal stability, and ability to be disintegrated upon demand by lowering the pH, we hypothesize that ZIF-8 can serve as a protective encapsulant for preserving the structure of various protein biomarkers in biofluids under unregulated temperatures. In this study, we introduce a novel technique for preserving protein biomarkers in biofluids (urine, serum, plasma, and blood), in dry state, by combining MOF-based preservation with dry spot sample collection. The technology introduced here involves simple mixing of biofluid samples with ZIF-8 precursors and drop-casting of a specific volume of the mixture onto a paper substrate to allow air-drying. This simple and easily deployable process results in encapsulation of protein biomarkers within ZIF-8 crystals that could be easily dissociated upon demand at a later time. The technique overcomes the drawbacks of conventional dry spot technology (i.e., limited stability of the dried specimen, the need for refrigeration and inaccuracy in determining the concentration of the biomarker) while retaining transportation and storage convenience (Figure 1). We choose neutrophil gelatinase-associated lipocalin (NGAL), a urinary biomarker for acute kidney injury,46,47 and CA-125, a serum/plasma biomarker for ovarian cancer48,49 as the model proteins. By using the commonly employed bioanalytical tools such as enzyme-linked immunosorbent assay (ELISA), circular dichroism spectroscopy, and fluorescent protein microarray, we demonstrate that the ZIF-8 encapsulation confers excellent structural stability to protein biomarkers 1292

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Figure 2. (A) Schematic illustrating the sample preparation of ZIF-8 encapsulated biofluid on a paper substrate. SEM image of a paper substrate after drying NGAL-spiked artificial urine (B) with and (C) without ZIF-8 precursors. (D) SEM image of NGAL-embedded ZIF-8 crystals showing uniform size and shape. Inset: TEM image of a typical NGAL-embedded ZIF-8 crystal. Scale bar: 200 nm. (E) XRD pattern of pure ZIF-8 and NGAL-embedded ZIF-8 crystals. (F) FTIR spectra of NGAL, pure ZIF-8, and NGAL-embedded ZIF-8 crystals. (G) Raman spectra of NGAL, pure ZIF-8, and NGAL-embedded ZIF-8 crystals.

(Figure S1) since the proteins are too small to be visible at this magnification. Owing to their rich functionality (such as carboxyl, carbonyl, hydroxyl, and imidazole groups), proteins serve as nucleation sites for the formation of ZIF-8 crystals.37 Here, we expected that ZIF-8 crystals would form and encapsulate NGAL in the presence of ZIF-8 precursors in the NGAL-spiked artificial urine. Indeed, the NGAL-embedded ZIF-8 crystals formed after 1 h incubation of the mixture solution and could be collected by centrifugation. The SEM and transmission electron microscopy (TEM) images show that the crystals formed in the NGAL-spiked artificial urine exhibit uniform size of ∼0.5 μm (Figure 2D), with a rhombic dodecahedral shape, similar to that of pure ZIF-8 crystals (Figure S2). The powder X-ray diffraction (XRD) pattern of the crystals also exhibits the typical peaks of pure ZIF-8 crystals (Figure 2E). To confirm the encapsulation of NGAL into ZIF-8 crystals, both pure ZIF-8 and NGAL-embedded ZIF-8 crystals were subjected to calcination (325 °C for 2 h). Only NGALembedded ZIF-8 showed pores in the calcinated crystals, indicating the encapsulation of protein in the ZIF-8 crystals (Figure S3). To further ascertain the formation of NGALembedded ZIF-8 crystals, Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy were employed. The FTIR spectrum obtained from the crystals not only shows typical ZIF-8 absorption peaks at 1584 cm−1 (CN stretching

in biospecimen in dry state, even when stored at nominal room temperature or high ambient temperatures encountered in different parts of the world. Such a biospecimen technology will not only alleviate huge financial and environmental burdens associated with “cold chain”, but also extend biomedical research benefits to underserved populations by acquiring clinical samples from regions/populations currently inaccessible.



RESULTS AND DISCUSSION As a proof-of-concept, NGAL-spiked artificial urine was employed as a model biospecimen. To prepare a typical ZIF8 preserved sample, NGAL-spiked artificial urine (50 μg/mL, 25 μL) was first mixed with 2-methylimidazole solution (640 mM, 12.5 μL) and then zinc acetate solution (160 mM, 12.5 μL) and incubated at room temperature for 1 h. Subsequently, the mixture (50 μL) was air-dried on a 0.5 × 2 cm2 Whatman 903 paper strip (which usually takes 2 h at room temperature) (Figure 2A). Scanning electron microscope (SEM) images show the distinct morphologies of paper substrates after drying NGAL-spiked artificial urine with and without ZIF-8 precursors (Figure 2B and 2C). The granular morphology of the paper substrate with artificial urine mixed with ZIF-8 precursors suggests the formation of ZIF-8 crystals (Figure 2B). Conversely, the morphology of the sample without adding ZIF-8 precursors (Figure 2C) is similar to that of bare paper 1293

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Figure 3. (A) Preservation efficacy of NGAL with and without ZIF-8 encapsulation. NGAL added on a paper card was stored at 25, 40, or 60 °C for 1 week. The ZIF-8 encapsulation shows comparable preservation, after storage at either room temperature or elevated temperatures, to the refrigeration method (freezing liquid samples at −20 °C). (B) Preservation efficacy of NGAL on paper card at 25, 40, or 60 °C for different durations. Results are the mean and standard deviation from three independent samples. (C) Preservation efficacy of NGAL in urine from three acute kidney injury patients with and without ZIF-8 encapsulation on paper cards stored at 25 or 40 °C for 1 week. Results are the mean and standard deviation from three independent samples. (D) Fluorescence intensity maps (generated from protein microarray) of #3 patient’s urinary biomarkers with and without ZIF-8 encapsulation on paper cards stored at 25 or 40 °C for 1 week. Frozen liquid sample (−20 °C) was used as a reference. (E) Plot depicting the fluorescence intensity of various biomarkers for the samples described in (D). POSs represent three distinct positive control signal intensities (POS1 > POS2 > POS3). Results are the mean and standard deviation from two parallel dots for each biomarker.

of imidazole) and 1400−1500 cm−1 (the imidazole ring stretching), but also exhibits absorption peaks at 1640−1670 and 1520−1560 cm−1, corresponding to amide I and amide II bands of protein, respectively (Figure 2F).50 In contrast, pure ZIF-8 crystals and NGAL only show their respective characteristic peaks. Similar results are observed by Raman spectroscopy, which also indicate the encapsulation of NGAL by ZIF-8 crystals (Figure 2G). The Raman spectrum obtained from pure NGAL exhibits a broad band at 1630−1690 cm−1, corresponding to amide I band of protein.51 The NGAL-encapsulating ZIF-8 crystals exhibit the amide I band of NGAL (not present in pure ZIF-8 crystals) and 2-methylimidazole characteristic bands at 1146, 1182, 1384, 1460, and 1505 cm−1 corresponding to C−N stretching, C−N stretching plus N−H wagging, CH3 bending, C−H wagging, and C−N stretching plus N−H wagging, respectively. 52 To quantify the encapsulation efficiency, the supernatant after crystals centrifugation was collected and remaining NGAL concentration was determined using sandwich enzyme-linked immunosorbent assay (ELISA). The encapsulation efficiency was found to be dependent on the concentration of ZIF-8 precursors. Specifically, when the concentrations of zinc acetate and 2-methylimidazole in the mixture increased to 40 mM and 160 mM, respectively, ∼95% NGAL was encapsulated within ZIF-8 crystals (Figure S4). As a control experiment, simple mixing of NGAL-spiked artificial

urine with pure ZIF-8 crystals resulted in extremely low (∼10%, owing to the physical adsorption) encapsulation efficiency. This physical mixing of preformed ZIF-8 crystals with the protein biomarkers is in stark contrast with the protein-embedding approach (i.e., formation of ZIF-8 crystals in the presence of protein biomarkers), which exhibited high encapsulation efficiency (95%). Next, we set out to evaluate the efficacy of ZIF-8 encapsulation in preserving NGAL upon exposure to harsh conditions (such as elevated temperatures) and accelerated stability testing that would normally lead to protein denaturation and loss of biospecimen integrity. NGAL-spiked artificial urine dried on paper substrates with and without ZIF-8 encapsulation were stored at 25, 40, or 60 °C for 1 week. Before analysis, the optimized elution buffer (0.2 M phosphate buffer at pH 5.6 + Tween 20 + EDTA, see Figures S5 and S6 for details) was used to elute NGAL from the paper substrates. The slightly acidic environment (at or below pH 6) was employed to dissociate ZIF-8 crystals and release encapsulated NGAL via breaking coordination between the zinc ions and imidazolate.27 We found that the ZIF-8 encapsulated NGAL dried on the paper was almost fully eluted (>95%) whereas NGAL alone dried on the paper was only partially eluted (∼75%) (Figure S6). It is important to note that ZIF-8 encapsulation prevented irreversible adsorption of proteins on 1294

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Figure 4. (A) Preservation efficacy of CA-125 spiked in serum with different dilutions. Storage condition: 40 °C for 1 week. (B) XRD patterns of ZIF-8 crystals formed in the serum with different dilutions. (C) SEM images of ZIF-8 crystals formed in 5- and 20-fold diluted serum. (D−F) Preservation efficacy of CA-125 spiked in serum, heparin-plasma or EDTA-plasma on paper card at 25, 40, or 60 °C for different durations. Results are the mean and standard deviation from three independent samples. Within 4 weeks, the ZIF-8 encapsulation shows comparable preservation, after storage at 25 and 40 °C, to the refrigeration method (freezing liquid samples at −20 °C).

the cellulose fibers by creating a crystal interface between the protein and paper substrate. We also confirmed that the slightly acidic elution buffer and the ZIF-8 residue did not alter the protein characteristics and downstream bioanalysis (Figure S7). After storage and elution, the concentration of NGAL in the eluate was quantified using the NGAL sandwich ELISA. The preservation efficacy (preservation %) was calculated by comparing the NGAL amount in the eluate to the spiked NGAL amount in the artificial urine. As shown in Figure 3A, there was more than 85% preservation of NGAL with ZIF-8 encapsulation after 1 week storage at 25 and 40 °C as well as more than 80% at 60 °C. Notably, at 25 and 40 °C, NGAL with ZIF-8 encapsulation showed comparable preservation to freeze−thawed liquid samples (the refrigeration approach as the control) stored at −20 °C. On the other hand, NGAL without ZIF-8 encapsulation stored at these temperatures for 1 week exhibited less than 30% preservation although 70% of the proteins were eluted as measured by bicinchoninic acid (BCA) assay, indicating the denaturation of NGAL under these storage conditions (Figure S8). To further confirm that ZIF-8 encapsulation preserves the encapsulated protein structure, circular dichroism (CD) spectroscopy was employed to characterize the secondary structure of human serum albumin (HSA) with and without ZIF-8 encapsulation after 1 week incubation at 40 °C (Figure S9). As expected, elevated temperature caused a significant change (a decrease in the αhelical content) of the secondary structure of unencapsulated HSA, as shown in the CD spectrum. In contrast, the secondary structure of ZIF-8 encapsulated HSA was found to be very similar to that of the pristine HSA, indicating that ZIF-8 encapsulation is able to preserve the structure of encapsulated protein.

We also found that the preservation efficacy critically depended on the concentrations of 2-methylimidazole and zinc acetate (Figure S10). A low preservation was noted upon using 40 mM 2-methylimidazole with 10 mM zinc acetate, which can be attributed to incomplete encapsulation of NGAL under this condition (∼70% encapsulation of NGAL using 40 mM 2-methylimidazole with 10 mM zinc acetate, Figure S4). Subsequently, using the optimal ZIF-8 precursor concentration (160 mM 2-methylimidazole and 40 mM zinc acetate), we extended the storage time under different temperatures up to 4 weeks (Figure 3B). Different dried paper strips were sampled at selected time points (2, 3, or 4 weeks) to monitor possible changes in NGAL preservation. With ZIF-8 encapsulation, over 70% of NGAL on paper was preserved up to 4 weeks (the maximum time tested) at all three temperatures (25, 40, and 60 °C), as opposed to 20% preservation on samples without ZIF-8 encapsulation. Notably, the ZIF-8 encapsulation had comparable preservation (∼90%, at 25 and 40 °C) to the freezing method over 2 weeks (−20 °C). Remarkably, 70% of NGAL on paper could be preserved within 4 weeks at 60 °C, which represented an extremely harsh storage condition (a surrogate for long-term storage stability at room temperature). Following the successful optimization of ZIF-8 encapsulation using spiked artificial urine samples, we set out to evaluate the applicability of this technique to patient urine samples. Three patients with acute kidney injury with different urinary NGAL levels (Patient #1, 32.8 ng/mL; Patient #2, 17.4 ng/mL; and Patient #3, 97.5 ng/mL; quantified by ELISA and confirmed by Western blotting, Figure S11) were selected. Compared to artificial urine, human urine is more complex due to the presence of numerous proteins such as albumin and globulins, solutes, and even whole cells (e.g., red blood cells and shed 1295

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Figure 5. (A) Procedure of preserving protein biomarkers in fresh blood samples by combining hand-powered centrifuge (“paperfuge”) and MOFbased preservation. (B) Preservation efficacy of CA-125 spiked in blood under temperature fluctuations in lab. Storage condition: 2 days 25 °C followed by 2 days 40 °C as one cycle and run for two cycles. (C) Preservation efficacy of CA-125 spiked in blood after 10 days of unknown shipping and handling conditions. The samples were mailed from Missouri to California and sent back to Missouri using regular mailing service. Results are the mean and standard deviation from three independent samples.

kidney cells).53 As routinely done, the cells were removed by low speed centrifugation before ZIF-8 encapsulation. We confirmed that mixing patient urine with ZIF-8 precursors resulted in a solution at near neutral pH. Thus, protein biomarkers in the mixture are not subjected to extremely low or high pH (Table S1). To assess the preservation efficacy of NGAL, the urine samples with and without ZIF-8 encapsulation dried on paper substrates were stored at 25 or 40 °C for 1 week. NGAL concentration in the eluate was quantified using ELISA. For all three patients, the samples with ZIF-8 encapsulation resulted in more than 85% NGAL preservation after 1 week storage at both 25 and 40 °C, whereas the control samples without ZIF-8 encapsulation showed less than 40% NGAL preservation (Figure 3C). To further demonstrate the capability of this technique to simultaneously preserve multiple biomarkers in patient samples, the recovered urine samples (with and without ZIF-8 encapsulation, after storage at 40 °C for 1 week) from Patient #3 were assayed by a multiplexed analysis tool, a protein microarray (Figure 3D,E). The frozenliquid samples stored at −20 °C were employed as the reference. The results showed that four detectable acute kidney injury biomarkers54,55 (albumin, NGAL, cystatin C, and beta-2 microglobulin) were almost fully (>95%) preserved with ZIF-8 encapsulation, whereas samples without ZIF-8 encapsulation exhibited ∼40−60% fluorescence signal intensity loss corresponding to these biomarkers. Overall, these results clearly demonstrate the efficacy of ZIF-8 encapsulation in preserving the dried urinary protein biomarkers on paper substrates at high temperatures.

Finally, we turned our attention to blood (and components serum and plasma), the most common biospecimens in biological and clinical studies. Compared to urine, serum or plasma represents a more complex biological matrix due to the presence of large amount of various proteins such as albumin, globulins and fibrinogen. Before proceeding to assess the preservation of specific protein biomarker, we confirmed that different types of proteins can be extracted from the paper substrates containing dried serum with or without ZIF-8 encapsulation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating the potential of this technique in preserving multiple protein biomarkers simultaneously in serum or plasma (Figure S12). CA-125, a serum biomarker for ovarian cancer,48 was used as the model protein and spiked into serum from healthy people. Considering extremely high concentration of serum proteins, the serum was first diluted (5-, 10- or 20-fold) before spiking CA-125 and adding ZIF-8 precursors to ensure ZIF-8 formation and more complete encapsulation. The CA-125 spiked serum samples with and without ZIF-8 encapsulation were dried on paper and stored at 40 °C for 1 week. The results indicated that CA-125 spiked in 20-fold diluted serum afforded the highest preservation (∼90%, Figure 4A). The poor preservation of CA-125 from 5- and 10-fold diluted serum was due to the incomplete ZIF-8 formation and encapsulation (Figure S13). This was further confirmed by XRD and SEM imaging (Figure 4B,C), revealing decreased ZIF-8 crystal formation with increased total protein concentration in serum. Subsequently, CA-125 was spiked into three different matrices that represent typical blood-derived biospecimens (serum, heparin-anticoagu1296

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under nonrefrigerated storage conditions. Using urinary NGAL and serum/plasma CA-125 as the model protein biomarkers, we demonstrate that the ZIF-8 encapsulation approach can preserve protein biomarkers in urine, serum, plasma, and blood at room temperature and 40 °C, with comparable or better preservation efficacy than the refrigeration method (freezing liquid samples at −20 °C). The protein can be recovered by dissociating the ZIF-8 protective layer in pH 6 buffer without affecting the protein structural integrity and downstream analysis. By combining the MOF-based preservation approach with dry spot sample collection method, the protein biomarker in biofluid can be preserved in dry state, greatly improving the biofluid-related biospecimen collection and handling capability in resource-limited settings. Overall, this energy-efficient and environmentally friendly approach not only represents a novel technique to eliminate the “cold chain” and temperaturecontrolled handling of biofluid-related biospecimens, but also allows interruptible, storable, and restorable on-demand analysis at a later time in a centralized or distributed location/manner to improve the reliability of the bioanalytical results. We believe that this facile and low-cost approach would open up the new avenues in both research and clinical settings such as large-scale cancer screening, epidemiologic studies from tropical and disaster-struck areas, remote chronic disease monitoring, and clinical trials for new drugs. We envision a “Biopreservation Kit” containing ZIF-8 precursors, paper strips, and transfer pipettes that would enable patients to self-prepare dried blood/urine samples and send to hospitals or clinical laboratories via regular mailing. Ultimately, this approach can alleviate hospital and logistical burdens, facilitate disease monitoring and patient feedback, and offer new services for currently underserved populations.

lated plasma and EDTA-anticoagulated plasma from healthy people with 20-fold dilution), dried on paper substrates with and without ZIF-8 encapsulation, and subsequently stored at 25, 40, or 60 °C for different durations of time. CA-125 concentration in the eluate was quantified using sandwich ELISA. In all three matrices, CA-125 with ZIF-8 encapsulation exhibited ∼85% preservation after 4 weeks storage at 25 and 40 °C as well as ∼75% at 60 °C (Figure 4D−F). On the other hand, CA-125 without ZIF-8 encapsulation stored at these three temperatures for 4 weeks displayed ∼50%, 60%, and 70% degradation, respectively. Remarkably, the preservation efficacy of ZIF-8 encapsulated CA-125 over 4 weeks of storage at 25 and 40 °C was comparable to the refrigeration method (freezing liquid samples at −20 °C) for the same storage duration. In the end, we set out to assess the applicability of this technique to preserve blood samples drawn in resource-limited settings. Unlike serum and plasma, the presence of a large quantity of whole cells (red and white blood cells and platelets) in blood may affect ZIF-8 encapsulation of target protein biomarkers. Considering that ZIF-8 can also form a thick layer on cell surfaces56 and lead to incomplete encapsulation or preservation of target protein biomarkers, it is important to remove blood cells through centrifugation before ZIF-8 encapsulation of protein biomarkers. Unfortunately, centrifuge is typically inaccessible in resource-limited settings considering that conventional systems are bulky, expensive, and electrically powered. Here, we implemented a hand-powered centrifuge introduced by Prakash’s group57 that was able to separate plasma from whole blood within 10 min for subsequent ZIF-8 encapsulation (Figure 5A). To mimic fresh whole blood samples from ovarian cancer patients, four different concentrations of CA-125 within the pathological-relevant range from 25 ng/mL-25 μg/mL were spiked into fresh blood from healthy volunteers.58,59 Then the plasma samples (heparin-anticoagulated) were separated from blood and diluted 20-fold for ZIF-8 encapsulation. To assess the preservation efficacy of this approach under unregulated conditions, two sets of experiments were devised. First, the ZIF-8 preserved plasma samples with the four different CA-125 concentrations were subjected to temperature fluctuations for 8 days (2 days 25 °C followed by 2 days 40 °C as one cycle and run for two cycles, Figure 5B). Second, the ZIF-8 preserved plasma samples with the four different CA-125 concentrations were shipped to California, USA and sent back to Missouri, USA via a regular shipping package (10 days in unknown shipping and handling conditions, Figure 5C). Unencapsulated samples dried on papers were used as negative controls in both cases. As shown in Figure 5B and C, samples with ZIF-8 encapsulation can achieve up to 90% of preservation, as opposed to ∼50−60% of preservation from control samples. It is also important to note that the preservation efficacy did not significantly change with the variation of CA-125 concentrations. Overall, the experiments here clearly demonstrate the feasibility and robustness of this approach in preserving protein biomarkers in blood samples. By combining with the hand-powered centrifuge, it is possible to directly collect and preserve fresh blood samples in resource-limited settings.



EXPERIMENTAL SECTION

Chemicals. 2-Methylimidazole, zinc acetate dihydrate, ethylenediaminetetraacetic acid (EDTA), Tween 20, sodium phosphate monobasic, and sodium phosphate dibasic were purchased from Sigma-Aldrich. Artificial urine (Surine Negative Urine Control) was purchased from Cerilliant Company (a Sigma-Aldrich Company). This artificial urine is suitable for LC−MS or GC−MS applications in clinical chemistry, urine drug testing, or forensic analysis. No protein and preservatives are added. Human recombinant neutrophil gelatinase-associated lipocalin (NGAL), human recombinant CA125, NGAL ELISA kit (DY1757, detection range: 78 pg/mL to 5000 pg/mL), and CA-125 ELISA kit (DY5609, detection range: 31.2 pg/ mL to 2000 pg/mL) were purchased from R&D systems. Custom protein microarray kits were purchased from RayBiotech (Custom GSeries Antibody Array, AAX-CUST-G). Pierce bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Fisher Scientific. For patient samples, approval was obtained from the Washington University Institutional Review Board, and written informed consent was obtained from all patients. Sample Preparation. NGAL-spiked artificial urine (50 μg/mL of NGAL, 25 μL) or patient urine (25 μL) was first mixed with 2methylimidazole aqueous solution (12.5 μL) and then zinc acetate dihydrate aqueous solution (12.5 μL). The final concentrations of 2methylimidazole after mixing were 320 mM, 160 mM, 80 mM, and 40 mM. The final concentrations of zinc acetate dihydrate after mixing were 80 mM, 40 mM, 20 mM, and 10 mM. Note that the molar ratio of 2-methylimidazole to zinc acetate dihydrate was kept at 4:1. After 1 h incubation at room temperature (20−23 °C), a 50 μL mixture was transferred onto a 2 × 0.5 cm2 Whatman 903 paper strip (Sigma) to allow air-drying (usually about 2 h at room temperature). To accurately determine the NGAL recovery after storage, it is important to avoid liquid leakage from the paper strip during the drop-cast



CONCLUSION In summary, we report a novel biospecimen preservation approach involving the use of a nanoporous material (ZIF-8) as an encapsulant for preserving protein biomarkers in biofluids 1297

DOI: 10.1021/acs.chemmater.7b04713 Chem. Mater. 2018, 30, 1291−1300

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Chemistry of Materials process. Typically, a 2 × 0.5 cm2 paper strip can absorb 50 μL of biofluid without leakage. After drying, the paper strips were sealed in Petri dishes and stored at 25, 40, or 60 °C for different time intervals. The sample preparation for serum and plasma were similar to urine except that the serum and plasma were first diluted 5-, 10-, or 20-times with PBS and then spiked with CA-125 (50 μg/mL of CA-125). For the fresh blood samples, the CA-125 was first spiked into the blood, and then the plasma was separated from the blood using handpowered centrifuge for the subsequent sample preparation. Protein Recovery. Before analysis, the paper strips were eluted in 1 mL of elution buffer (0.2 M phosphate buffer with 2 mM EDTA and 0.1% Tween 20 at pH = 5.6) by shaking the paper strip in a cuvette with the elution buffer at the speed of 60 rpm for 1 h. Different elution buffer recipes were tested for maximal recovery (Figures S5 and S6). The elution solution was then assayed by ELISA for the target analyte. NGAL and CA-125 standards provided with the ELISA kits were used to generate a standard curve for each assay. Characterization. To characterize protein-embedded crystals, crystals were centrifuged and washed with DI water twice (8000 rpm for 20 min). To calculate the encapsulation efficiency, the supernatant after first centrifugation was collected and assayed by ELISA. SEM images were obtained using a FEI Nova 2300 fieldemission scanning electron microscope at an acceleration voltage of 10 kV. Fourier transform infrared spectroscopy (FTIR) measurements were conducted using a Nicolette Nexus 470 spectrometer. The Raman spectra were obtained using a Renishaw inVia confocal Raman spectrometer mounted on a Leica microscope with a 50× objective and a 514 nm wavelength diode laser as an illumination source. The Xray diffraction (XRD) measurements of the samples were recorded on a Bruker D8-Advance X-ray powder diffractometer using Cu Kα radiation (λ = 1.5406 Å). SDS-PAGE and Western Blotting Protocols. For SDS-PAGE, 300 μL of eluate was mixed with 0.9 mL of acetone−methanol (1:1), placed on ice for 1 h, and centrifuged at 10 000 rpm for 10 min to precipitate and concentrate the eluted urine or serum proteins. The protein pellet was briefly air-dried and resuspended in SDS sample buffer containing 5% mercaptoethanol. A 5 μL sample was applied to each well of a NuPAGE 4−12% acrylamide Bis-Tris gel (Invitrogen, San Diego, CA). The proteins were separated at a constant 200 V for 35 min using MES running buffer. The gel was stained with 0.1% coomassie brilliant blue solution for 3 h and then destained overnight. For Western blotting of patient urine samples, 100 μL of thawed urine was mixed with 1 mL of acetone−methanol (1:1) and placed on ice for 1 h for precipitation, gathered by centrifugation, and resuspended in SDS sample buffer containing 5% mercaptoethanol. After SDS-PAGE, the urine proteins and prestained molecular weight markers were transferred to nitrocellulose membranes for 6 min using iBLOT (Invitrogen). The nitrocellulose membrane was briefly washed with water and the nonspecific sites blocked with LI-COR block solution (LI-COR Biosciences, Lincoln, NE). Urinary albumin and NGAL were visualized by incubation with 1/2000 dilution of rabbit antihuman serum albumin (Abcam, Cambridge, UK) and 1/500 dilution of goat antihuman NGAL (R&D Systems, Minneapolis, MN) in LI-COR block buffer containing 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO) overnight. The membrane was then washed three times with phosphate-buffered saline containing 0.05% Tween-20 followed by incubation with 1/10 000 dilutions each of Donkey antirabbit IgG 680 and Donkey antigoat IgG 800 (LI-COR Biosciences). After 1 h, the membrane was washed four times with phosphate-buffered saline containing 0.05% Tween-20 and visualized using an Odyssey-Fc (LICOR Biosciences). Protein Microarray Protocols. Commercial protein microarray chip was purchased from RayBiotech (Custom G-Series Antibody Array, AAX-CUST-G). Capture antibodies were printed on a glass slide with four subarrays available per slide. The slide was blocked by 1× blocking buffer (0103004-B) for 30 min. The eluted urine samples were added into each subwell of the microarray chip for 2 h incubation at room temperature. The chip was then washed thoroughly with 1× wash buffers (0103004). Seventy microliters of 1× biotin-conjugated detection antibodies was added to each subarray, and the chip was

incubated in room temperature with gentle shaking. After 2 h, the chip was washed, and 70 μL of streptavidin-CW800 (100 ng/mL in 1× blocking buffer) was added and incubated in dark for 20 min. The chip was washed thoroughly with wash buffer then nanopure water and blow dried under nitrogen gas. The glass chip was scanned by Licor Odyssey CLx scanner using 800 nm channel (intensity = 2, resolution = 21 μm, scanning height = 1 mm). Median background signal was adopted for analysis spot intensity. CD Spectroscopy. The CD measurements were performed using a spectropolarimeter JASCO J-810. The spectrum was collected at the rate of 20 nm per minute at a response time of 16 s. Before CD measurement, the ZIF-8 encapsulated HSA was first eluted and then filtered to remove any ZIF-8 byproduct using centrifuge tube with 30 kDa filter. The HSA recovered from various treatments was quantified by BCA assay for calculating molar ellipticity. The secondary structures of HSA (α-helical content, β-sheet content) were analyzed using CDPro software from CD spectra. Paperfuge. The paperfuge was composed of a paper disc, a string, and glass capillaries (microhematocrit capillary tubes, D = 1.55 mm, Fisher Scientific). Common wood was used for the handles. The string was immobilized through paper disc using epoxy. After blood was drawn, one end of capillary was sealed by capillary tube sealing tray (Thomas Scientific).



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b04713. SEM images of bare paper and pure ZIF-8; TEM images of calcinated ZIF-8; study of encapsulation, elution, and preservation efficiency; protein quantification with BCA assay and ELISA; CD experiment for studying protein structure; Western blot of patient samples; SDS-PAGE of eluates from dried serum samples; study of patient urine pH after mixing with ZIF-8 precursors (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jeremiah J. Morrissey: 0000-0002-9911-4811 Srikanth Singamaneni: 0000-0002-7203-2613 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We acknowledge support from Air Force Office of Scientific Research (FA9550-15-1-0228) and National Institutes of Health (R01 CA141521). The authors thank the Nano Research Facility (NRF) at Washington University for providing access to electron microscopy facilities.



REFERENCES

(1) Ellervik, C.; Vaught, J. Preanalytical Variables Affecting the Integrity of Human Biospecimens in Biobanking. Clin. Chem. 2015, 61, 914−934. (2) Moore, H. M.; Kelly, A.; Jewell, S. D.; McShane, L. M.; Clark, D. P.; Greenspan, R.; Hayes, D. F.; Hainaut, P.; Kim, P.; Mansfield, E.; Potapova, O.; Riegman, P.; Rubinstein, Y.; Seijo, E.; Somiari, S.; Watson, P.; Weier, H.-U.; Zhu, C.; Vaught, J. Biospecimen Reporting for Improved Study Quality (BRISQ). J. Proteome Res. 2011, 10, 3429−3438.

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Transmission Electron Microscopy. J. Am. Chem. Soc. 2015, 137, 7322−7328. (23) Murray, L. J.; Dinca, M.; Long, J. R. Hydrogen Storage in Metalorganic Frameworks. Chem. Soc. Rev. 2009, 38, 1294−1314. (24) Cao, X.; Tan, C.; Sindoro, M.; Zhang, H. Hybrid Micro-/nanostructures Derived from Metal-organic Frameworks: Preparation and Applications in Energy Storage and Conversion. Chem. Soc. Rev. 2017, 46, 2660−2677. (25) Horcajada, P.; Serre, C.; Vallet-Regi, M.; Sebban, M.; Taulelle, F.; Ferey, G. Metal-organic Frameworks as Efficient Materials for Drug Delivery. Angew. Chem., Int. Ed. 2006, 45, 5974−5978. (26) Wu, M.-X.; Yang, Y.-W. Metal−Organic Framework (MOF)Based Drug/Cargo Delivery and Cancer Therapy. Adv. Mater. 2017, 29, 1606134. (27) Zheng, H.; Zhang, Y.; Liu, L.; Wan, W.; Guo, P.; Nyström, A. M.; Zou, X. One-pot Synthesis of Metal−Organic Frameworks with Encapsulated Target Molecules and Their Applications for Controlled Drug Delivery. J. Am. Chem. Soc. 2016, 138, 962−968. (28) Ma, L. Q.; Abney, C.; Lin, W. B. Enantioselective Catalysis with Homochiral Metal-organic Frameworks. Chem. Soc. Rev. 2009, 38, 1248−1256. (29) Yu, Y.; Wu, X.-J.; Zhao, M.; Ma, Q.; Chen, J.; Chen, B.; Sindoro, M.; Yang, J.; Han, S.; Lu, Q.; Zhang, H. Anodized Aluminum Oxide Templated Synthesis of Metal−Organic Frameworks Used as Membrane Reactors. Angew. Chem., Int. Ed. 2017, 56, 578−581. (30) Zhu, H.; Yang, X.; Cranston, E. D.; Zhu, S. Flexible and Porous Nanocellulose Aerogels with High Loadings of Metal−OrganicFramework Particles for Separations Applications. Adv. Mater. 2016, 28, 7652−7657. (31) Chen, Y.; Zhang, S.; Cao, S.; Li, S.; Chen, F.; Yuan, S.; Xu, C.; Zhou, J.; Feng, X.; Ma, X.; Wang, B. Roll-to-Roll Production of MetalOrganic Framework Coatings for Particulate Matter Removal. Adv. Mater. 2017, 29, 1606221. (32) Kreno, L. E.; Leong, K.; Farha, O. K.; Allendorf, M.; Van Duyne, R. P.; Hupp, J. T. Metal-Organic Framework Materials as Chemical Sensors. Chem. Rev. 2012, 112, 1105−1125. (33) Furukawa, H.; Cordova, K. E.; O’Keeffe, M.; Yaghi, O. M. The Chemistry and Applications of Metal-Organic Frameworks. Science 2013, 341, 1230444. (34) Foo, M. L.; Matsuda, R.; Kitagawa, S. Functional Hybrid Porous Coordination Polymers. Chem. Mater. 2014, 26, 310−322. (35) Lu, G.; Farha, O. K.; Zhang, W. N.; Huo, F. W.; Hupp, J. T. Engineering ZIF-8 Thin Films for Hybrid MOF-Based Devices. Adv. Mater. 2012, 24, 3970−3974. (36) Wang, Q.; Feng, X.; Wang, S.; Song, N.; Chen, Y.; Tong, W.; Han, Y.; Yang, L.; Wang, B. Metal-Organic Framework Templated Synthesis of Copper Azide as the Primary Explosive with Low Electrostatic Sensitivity and Excellent Initiation Ability. Adv. Mater. 2016, 28, 5837−5843. (37) Liang, K.; Ricco, R.; Doherty, C. M.; Styles, M. J.; Bell, S.; Kirby, N.; Mudie, S.; Haylock, D.; Hill, A. J.; Doonan, C. J.; Falcaro, P. Biomimetic Mineralization of Metal-organic Frameworks as Protective Coatings for Biomacromolecules. Nat. Commun. 2015, 6, 7240. (38) Lyu, F. J.; Zhang, Y. F.; Zare, R. N.; Ge, J.; Liu, Z. One-Pot Synthesis of Protein-Embedded Metal-Organic Frameworks with Enhanced Biological Activities. Nano Lett. 2014, 14, 5761−5765. (39) Zhuang, J.; Young, A. P.; Tsung, C.-K. Integration of Biomolecules with Metal−Organic Frameworks. Small 2017, 13, 1700880. (40) Li, P.; Moon, S. Y.; Guelta, M. A.; Harvey, S. P.; Hupp, J. T.; Farha, O. K. Encapsulation of a Nerve Agent Detoxifying Enzyme by a Mesoporous Zirconium Metal-Organic Framework Engenders Thermal and Long-Term Stability. J. Am. Chem. Soc. 2016, 138, 8052− 8055. (41) Shieh, F. K.; Wang, S. C.; Yen, C. I.; Wu, C. C.; Dutta, S.; Chou, L. Y.; Morabito, J. V.; Hu, P.; Hsu, M. H.; Wu, K. C. W.; Tsung, C. K. Imparting Functionality to Biocatalysts via Embedding Enzymes into Nanoporous Materials by a de Novo Approach: Size-Selective

(3) Solivio, M. J.; Less, R.; Rynes, M. L.; Kramer, M.; Aksan, A. Adsorbing/dissolving Lyoprotectant Matrix Technology for Noncryogenic Storage of Archival Human Sera. Sci. Rep. 2016, 6, 24186. (4) Schrohl, A. S.; Wurtz, S.; Kohn, E.; Banks, R. E.; Nielsen, H. J.; Sweep, F.; Brunner, N. Banking of Biological Fluids for Studies of Disease-associated Protein Biomarkers. Mol. Cell. Proteomics 2008, 7, 2061−2066. (5) Chaigneau, C.; Cabioch, T.; Beaumont, K.; Betsou, F. Serum Biobank Certification and the Establishment of Quality Controls for Biological Fluids: Examples of Serum Biomarker Stability after Temperature Variation. Clin. Chem. Lab. Med. 2007, 45, 1390−1395. (6) Betsou, F.; Barnes, R.; Burke, T.; Coppola, D.; DeSouza, Y.; Eliason, J.; Glazer, B.; Horsfall, D.; Kleeberger, C.; Lehmann, S.; Prasad, A.; Skubitz, A.; Somiari, S.; Gunter, E. Human Biospecimen Research: Experimental Protocol and Quality Control Tools. Cancer Epidemiol., Biomarkers Prev. 2009, 18, 1017−1025. (7) Evans, M. J.; Livesey, J. H.; Ellis, M. J.; Yandle, T. G. Effect of Anticoagulants and Storage Temperatures on Stability of Plasma and Serum Hormones. Clin. Biochem. 2001, 34, 107−112. (8) Hubel, A.; Aksan, A.; Skubitz, A. P. N.; Wendt, C.; Zhong, X. State of the Art in Preservation of Fluid Biospecimens. Biopreserv. Biobanking 2011, 9, 237−244. (9) Shabihkhani, M.; Lucey, G. M.; Wei, B. W.; Mareninov, S.; Lou, J. J.; Vinters, H. V.; Singer, E. J.; Cloughesy, T. F.; Yong, W. H. The Procurement, Storage, and Quality Assurance of Frozen Blood and Tissue Biospecimens in Pathology, Biorepository, and Biobank Settings. Clin. Biochem. 2014, 47, 258−266. (10) Lieberman, D.; McClure, E.; Harston, S.; Madan, D. Maintaining Semen Quality by Improving Cold Chain Equipment used in Cattle Artificial Insemination. Sci. Rep. 2016, 6, 28108. (11) Mueller, C.; Edmiston, K. H.; Carpenter, C.; Gaffney, E.; Ryan, C.; Ward, R.; White, S.; Memeo, L.; Colarossi, C.; Petricoin, E. F.; Liotta, L. A.; Espina, V. One-Step Preservation of Phosphoproteins and Tissue Morphology at Room Temperature for Diagnostic and Research Specimens. PLoS One 2011, 6, e23780. (12) Delanghe, J.; Speeckaert, M. Preanalytical Requirements of Urinalysis. Biochem. Med. 2014, 24, 89−104. (13) Lippi, G.; Becan-McBride, K.; Behulova, D.; Bowen, R. A.; Church, S.; Delanghe, J.; Grankvist, K.; Kitchen, S.; Nybo, M.; Nauck, M.; Nikolac, N.; Palicka, V.; Plebani, M.; Sandberg, S.; Simundic, A. M. Preanalytical Quality Improvement: in Quality We Trust. Clin. Chem. Lab. Med. 2013, 51, 229−241. (14) Less, R.; Boylan, K. L. M.; Skubitz, A. P. N.; Aksan, A. Isothermal vitrification methodology development for non-cryogenic storage of archival human sera. Cryobiology 2013, 66, 176−185. (15) Bakaltcheva, I.; O’Sullivan, A. M.; Hmel, P.; Ogbu, H. Freezedried Whole Plasma: Evaluating Sucrose, Trehalose, Sorbitol, Mannitol and Glycine as Stabilizers. Thromb. Res. 2007, 120, 105−116. (16) Kluge, J. A.; Li, A. B.; Kahn, B. T.; Michaud, D. S.; Omenetto, F. G.; Kaplan, D. L. Silk-based Blood Stabilization for Diagnostics. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 5892−5897. (17) Frokjaer, S.; Otzen, D. E. Protein Drug Stability: A Formulation Challenge. Nat. Rev. Drug Discovery 2005, 4, 298−306. (18) Singh, S. K.; Kolhe, P.; Mehta, A. P.; Chico, S. C.; Lary, A. L.; Huang, M. Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding. Pharm. Res. 2011, 28, 873−885. (19) Iannuzzi, C.; Adrover, M.; Puglisi, R.; Yan, R.; Temussi, P. A.; Pastore, A. The Role of Zinc in the Stability of the Marginally Stable IscU Scaffold Protein. Protein Sci. 2014, 23, 1208−1219. (20) Dunn, M. F. Zinc-ligand Interactions Modulate Assembly and Stability of the Insulin Hexamer - A Review. BioMetals 2005, 18, 295− 303. (21) Yaghi, O. M.; O’Keeffe, M.; Ockwig, N. W.; Chae, H. K.; Eddaoudi, M.; Kim, J. Reticular Synthesis and the Design of New Materials. Nature 2003, 423, 705−714. (22) Patterson, J. P.; Abellan, P.; Denny, M. S.; Park, C.; Browning, N. D.; Cohen, S. M.; Evans, J. E.; Gianneschi, N. C. Observing the Growth of Metal−Organic Frameworks by in Situ Liquid Cell 1299

DOI: 10.1021/acs.chemmater.7b04713 Chem. Mater. 2018, 30, 1291−1300

Article

Chemistry of Materials Sheltering of Catalase in Metal-Organic Framework Microcrystals. J. Am. Chem. Soc. 2015, 137, 4276−4279. (42) Liao, F. S.; Lo, W. S.; Hsu, Y. S.; Wu, C. C.; Wang, S. C.; Shieh, F. K.; Morabito, J. V.; Chou, L. Y.; Wu, K. C. W.; Tsung, C. K. Shielding against Unfolding by Embedding Enzymes in Metal-Organic Frameworks via a de Novo Approach. J. Am. Chem. Soc. 2017, 139, 6530−6533. (43) Wang, C.; Tadepalli, S.; Luan, J.; Liu, K.-K.; Morrissey, J. J.; Kharasch, E. D.; Naik, R. R.; Singamaneni, S. Metal-Organic Framework as a Protective Coating for Biodiagnostic Chips. Adv. Mater. 2017, 29, 1604433. (44) Doonan, C.; Riccò, R.; Liang, K.; Bradshaw, D.; Falcaro, P. Metal−Organic Frameworks at the Biointerface: Synthetic Strategies and Applications. Acc. Chem. Res. 2017, 50, 1423−1432. (45) Jiang, Q.; Chandar, Y. J.; Cao, S.; Kharasch, E. D.; Singamaneni, S.; Morrissey, J. J. Rapid, Point-of-Care, Paper-Based Plasmonic Biosensor for Zika Virus Diagnosis. Adv. Biosyst 2017, 1, 1700096. (46) Devarajan, P. Review: Neutrophil Gelatinase-associated Lipocalin: A troponin-like Biomarker for Human Acute Kidney Injury. Nephrology 2010, 15, 419−428. (47) Abbas, A.; Tian, L. M.; Morrissey, J. J.; Kharasch, E. D.; Singamaneni, S. Hot Spot-Localized Artificial Antibodies for LabelFree Plasmonic Biosensing. Adv. Funct. Mater. 2013, 23, 1789−1797. (48) Gupta, D.; Lis, C. G. Role of CA125 in Predicting Ovarian Cancer Survival - A Review of the Epidemiological Literature. J. Ovarian Res. 2009, 2, 13. (49) Armstrong, D. K. Relapsed Ovarian Cancer: Challenges and Management Strategies for a Chronic Disease. Oncologist 2002, 7, 20− 28. (50) Hu, Y.; Kazemian, H.; Rohani, S.; Huang, Y.; Song, Y. In Situ High Pressure Study of ZIF-8 by FTIR Spectroscopy. Chem. Commun. 2011, 47, 12694−12696. (51) Yu, N.-T.; Liu, C. S.; O’Shea, D. C. Laser Raman Spectroscopy and the Conformation of Insulin and Proinsulin. J. Mol. Biol. 1972, 70, 117−132. (52) Kumari, G.; Jayaramulu, K.; Maji, T. K.; Narayana, C. Temperature Induced Structural Transformations and Gas Adsorption in the Zeolitic Imidazolate Framework ZIF-8: A Raman Study. J. Phys. Chem. A 2013, 117, 11006−11012. (53) Racusen, L. C. Epithelial Cell Shedding in Acute Renal Injury. Clin. Exp. Pharmacol. Physiol. 1998, 25, 273−275. (54) Herrero-Morín, J. D.; Málaga, S.; Fernández, N.; Rey, C.; Diéguez, M. Á .; Solís, G.; Concha, A.; Medina, A. Cystatin C and Beta2-microglobulin: Markers of Glomerular Filtration in Critically Ill Children. Crit. Care 2007, 11, R59. (55) Bolisetty, S.; Agarwal, A. Urine Albumin as a Biomarker in Acute Kidney Injury. Am. J. Physiol. Renal Physiol. 2011, 300, F626−F627. (56) Liang, K.; Richardson, J. J.; Cui, J. W.; Caruso, F.; Doonan, C. J.; Falcaro, P. Metal-Organic Framework Coatings as Cytoprotective Exoskeletons for Living Cells. Adv. Mater. 2016, 28, 7910−7914. (57) Bhamla, M. S.; Benson, B.; Chai, C.; Katsikis, G.; Johri, A.; Prakash, M. Hand-powered Ultralow-cost Paper Centrifuge. Nat. Biomed. Eng. 2017, 1, 0009. (58) Yin, B. W. T.; Lloyd, K. O. Molecular Cloning of the CA125 Ovarian Cancer Antigen - Identification as a New Mucin, MUC16. J. Biol. Chem. 2001, 276, 27371−27375. (59) Lenhard, M. S.; Nehring, S.; Nagel, D.; Mayr, D.; Kirschenhofer, A.; Hertlein, L.; Friese, K.; Stieber, P.; Burges, A. Predictive Value of CA 125 and CA 72−4 in Ovarian Borderline Tumors. Clin. Chem. Lab. Med. 2009, 47, 537−542.

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